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Master Thesis

Optimization of the in vivo biotinylation technology for thediscovery of accessible tissue-specific markers

Author(s): Pfaffen, Stefanie

Publication Date: 2006

Permanent Link: https://doi.org/10.3929/ethz-a-005185782

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Departement Chemie und Angewandte

Biowissenschaften

Institut Pharmazeutische Wissenschaften

Optimization of the in vivo biotinylation technology for the discovery of accessible tissue-specific markers

Diplomarbeit von

Stefanie Pfaffen Bürgerin von Mund (VS)

Leiter: Prof. Dr. Dario Neri

Betreuer: Jascha-Nikolai Rybak

ACKNOWLEDGMENTS The work described in this diploma thesis has been carried out in the laboratory and

under supervision of Prof. Dr. Neri at the Institute of Pharmaceutical Sciences of

ETH, Zürich. I would like to thank Prof. D. Neri for giving me the opportunity to work

in his group during my diploma thesis on the topic of in vivo biotinylation technology.

Special thanks go to Jascha Rybak for having me introduced into the laboratory

techniques needed for the realisation of this work. He was always there to support,

encourage and help me. All the perfusion experiments were carried out together with

Jascha Rybak.

Furthermore, I would like to express my gratitude to Simone Scheurer, Giuliano Elia,

Simon Brack, Patrizia Alessi, Jörg Scheuermann and Christoph Dumelin. They all

provided me with helpful advises, many hinds and tricks and spent their time for

helping me. The mass spectrometric experiments were carried out with Jascha

Rybak and Giuliano Elia.

I would also thank to all my colleagues of the group for providing me with a pleasant

working environment and for their direct and indirect assistence during my work.

Last, but not least, I would like to use this opportunity to say many thanks to my

parents and my boyfriend for believing in me and for supporting me during my studies

in Zürich.

I

ABSTRACT One avenue towards the increase of drug efficacy is the selective delivery of

therapeutic agents to the pathological tissue (diseased organ, tumor) by means of

their association to ligands endowed with high affinity for target proteins, specifically

expressed only in the diseased tissue and virtually absent in all other healthy organs.

Aiming at the discovery of novel tissue-specific targets accessible from circulation,

Rybak et al. have set up in our laboratory a novel technology – termed “in vivo

biotinylation”. This approach is based on the terminal perfusion of rodents with a

charged, activated ester derivative of biotin (Sulfo-NHS-LC-Biotin), which cannot

diffuse through biological membranes. Proteins, glycolipids and phospholipids

carrying accessible primary amino groups may be labeled by this procedure.

Biotinylated proteins can efficiently be purified on streptavidin resins and submitted to

a comparative proteomic analysis.

In the diploma thesis presented here, several approaches were attempted to

advance and optimize the in vivo biotinylation technology.

As the perfusion of tumours was much less efficient and more heterogeneous

compared to other organs, we set out to improve the perfusion of tumours by

introducing several modifications to the perfusion protocol. Omission of the PBS

wash prior to biotinylation, addition of dextran to the perfusion solutions, pre-warming

of the perfusion solutions and warming of the animal during anesthesia and perfusion

led to a much higher proportion of successfully biotinylated tumours.

A gel-based proteomic analysis led to the first identification of proteins labelled in the

in vivo biotinylation, among which the kidney-specific cadherin 16 was found to be

the most abundant accessible protein in rodent kidneys. However, the gel-based

proteomic analysis suffered from poor sensitivity. Two different approaches to

increase sensitivity failed.

II

III

In a gel-free proteomic shot-gun analysis Giuliano Elia identified up to more than

hundred proteins labelled in different organs during in vivo biotinylation.

A reduction of sample complexity by pre-fractionation of the protein extracts was

attempted, in order to improve the gel-free proteomic analysis and to lead to more

protein identifications by mass spectrometry. Therefore, a hydroxyapatite-based

chromatography method for the pre-fractionation of the protein extracts from mice

perfused with a biotinylation reagent was set up.

In the gel-free proteomic analysis of samples from in vivo biotinylated mice, besides

the expected proteins from cell plasma membrane and extracellular matrix, several

intracellular proteins were identified. To overcome this problem, Sulfo-NHS-SS-Biotin

was used as an alternative biotinylation reagent. However, much less biotinylated

proteins could be recovered and the proportion of intracellular proteins was not

reduced.

In a second approach, the synthesis of a dextran-biotin-NHS derivative was set up, in

order to reduce cytoplasmatic contaminants. A commercially available dextran

modified with lysins and biotins was reacted with a 100 x excess of disuccinimidyl

tartrate (DST). Excessive DST could successfully be eliminated through PD-10

chromatography. However, results from an activity test with Fluoresceinamine led to

the conclusion that only a low fraction of primary amines can be incorporated into the

synthesised dextran-NHS derivative.

In conclusion, this thesis contributed to a better in vivo biotinylation of tumours and

yielded a hydroxyapatite-based chromatography protocol for the pre-fractionation of

the proteins labelled in the in vivo biotinylation procedure.

ABBREVATIONS

BSA Bovin serum albumin

1D One-dimensional

2D Two-dimensional

2D-PAGE Two-dimensional polyacrylamide gel electrophoresis

DMEM Dulbecco’s Modified Eagle’s Medium

DTE Dithioerythritol

EDTA Ethylene diamine tetraacetic acid

FCS Foetal bovine serum

FPLC Fast performance liquid chromatography

FT-ICR Fourier transform in cyclotron

GSSG oxidized Glutathione

HA chromatography Hydroxyapatite chromatography

HAP-HPLC Hydroxyapatite high pressure liquid chromatography

HPLC High pressure liquid chromatography

HRP Horse radish peroxidase

kDa Kilo Dalten

LC-MS/MS Micro-liquid capillary tandem mass spectrometry

MALDI-TOF Matrix assisted laser desorption ionization-time of flight

MilliQ Millipore filtered water

MOPS 3-(N-morpholino)propane sulfonic acid

MS Mass spectrometry

MS/MS Tandem mass spectrometry

MW Molecular weight

MWCO Molecular weight cut off

PAGE Polyacrylamide gel electrophoresis

PB Phospate buffered solution

PBS Phospate buffered saline

rpm Revolutions per minute

RP-HPLC Reverse phase high pressure liquid chromatography

RT Room temperature

SA Streptavidin blot

SDS Sodium dodecyl sulfate

IV

V

TCA Trifluor chlor acid

TFA Trifluor acetic acid

Tris Tris(hydroxymethyl)aminomethane

Tween 20 Polyoxyethylene-sorbitan monolaurate

V Volume

CONTENTS ACKNOWLEDGMENTS I ABSTRACT II ABBREVIATIONS IV CONTENTS VI 1 INTRODUCTION 1

1.1 Tissue targeting 1 1.1.1 Tumour targeting 2

1.2 Discovery of novel accessible tissue-specific targets 5 1.2.1 In vitro model systems for the discovery of novel markers of

neovasculature 6 1.2.2 In vivo and ex vivo model systems for the identification of

vascular targets 7 1.2.3 Proteomic technologies for target identification 8

1.3 In vivo biotinylation technology 18 1.4 Experimental overview and aims of this diploma thesis 19

1.4.1 Optimization of the tumour perfusion 21 1.4.2 Optimization of the gel-based proteomic analysis 23 1.4.3 Optimization of the gel-free proteomic analysis 24 1.4.4 Alternative perfusion reagents 25

2 MATERIALS AND METHODS 27

2.1 Materials 27 2.1.1 Chemicals and reagents 27 2.1.2 Laboratory materials 33 2.1.3 Instruments 37 2.1.4 Solutions and buffers 41

2.2 Methods 42 2.2.1 Implantation of F9 teratocarcinoma allografts in SvEv129 mice 42 2.2.2 In vivo biotinylation 45 2.2.3 Perfusion and injection of mice with Hoechst dye 51 2.2.4 Histochemistry 52 2.2.5 Preparation of protein extracts from tissue specimens 54 2.2.6 Purification of biotinylated proteins 56 2.2.7 Sample concentration 59 2.2.8 Protein fractionation by hydroxyapatite chromatography 61 2.2.9 SDS-PAGE and Streptavidin blot 67 2.2.10 Protein visualisation 70 2.2.11 Identification of proteins 72

VI

2.2.12 Synthesis of a dextran-biotin-NHS derivative 83

3 RESULTS 87

3.1 Optimization of the in vivo biotinylation of tumours 87 3.1.1 Perfusion with a higher flow rate 88 3.1.2 Perfusion versus intravenous injection of mice with Hoechst dye 91 3.1.3 In vivo perfusion with an optimized protocol 93

3.2 Gel-based proteomic analysis 97 3.2.1 Purification of biotinylated proteins on streptavidin 97 3.2.2 Excision of gel bands and protein identification by LC-MS/MS 99 3.2.3 Optimization approach I: Protein concentration on resin 102 3.2.4 Optimization approach II: Sample concentration by spin dialysis 105

3.3 Fractionation of in vivo biotinylated proteins by hydroxyapatite chromatography for the optimization of the gel-free proteomic analysis 109 3.3.1 Fractionation of a standard protein mixture by hydroxyapatite

chromatography 109 3.3.2 Fractionation of a kidney protein extract from an in vivo

biotinylated mouse 114 3.3.3 Fractionation of a protein extract of a RENCA-bearing kidney

from an in vivo biotinylated mouse 118

3.4 In vivo biotinylation of tumor-bearing mice with Sulfo-NHS-SS- Biotin as an alternative biotinylation reagent 128

3.5 Synthesis of a dextran-biotin-NHS derivative as an alternative, less cell penetrating perfusion reagent 133 3.5.1 Pilot experiment: Elimination of the crosslinker simulated

by Fluorescein 133 3.5.2 Synthesis, purification and activity test of the dextran-biotin-

NHS-derivative 142 4 DISCUSSION 147

4.1 Perfusion of tumours 148 4.2 Gel-based proteomic analysis 153

4.3 Gel-free proteomic analysis 154

4.4 Pre-fractionation by hydroxyapatite chromatography 155

4.5 Alternative biotinylation reagents 157

VII

VIII

5 REFERENCES 160 6 CURRICULUM VITAE 169

1 INTRODUCTION 1.1 Tissue targeting The selective delivery of a therapeutic agent to a certain tissue could increase the

therapeutic index of a drug as the local concentration of the agent at the site of

disease would be increased, while at the same time site effects at other tissues could

be reduced. The identification of tissue-specific targets may represent the basis for

the development of a tissue-specific binder for selective drug delivery.

The molecular targeting of markers of disease only expressed in pathological

conditions, should allow both a more effective imaging and possibly a therapy. [Phil

Oh et al., 2004]. For example, radioimmunoguided surgery (RIGS) appears as an

efficient tool for accurate tumour detection up to the level of micrometastases by

detecting radiolabeled antibody-bound tumour cells during operation [Kim et al.,

2004]. Furthermore, radioimmunotherapy (RIT) strategies, in which, monoclonal

antibodies directed against tumour-specific antigens are used to deliver therapeutic

radioisotopes to sites of disseminated disease, could be used to target cancer. This

strategy has the advantage that cells adjacent to the structure to which an antibody

has bound could be killed through a “cross-fire” effect.

A major focus of research on marker discovery and selective drug delivery continues

to be in the area of tumour diagnosis and therapy. The incidence of cancer has

continued to grow over the last 30 years [Pratt et al., 1994] and cancer is today one

of the most threatening diseases in industrial countries. Each 3rd Western European

suffers from a tumour during his life [Pratt et al., 1994]. Complete remission of

tumours depends much on the time point of diagnosis and the tumour type.

Conventional cancer therapy goes along with severe side effects while often

providing only poor prognosis.

1

1.1.1 Tumour targeting 1.1.1.1 Classical tumour therapy Classical therapy of solid tumours consists of their surgical removal combined with

irradiation or chemotherapy. Following this line of therapy, new cytotoxic agents like

the taxanes (Paclitaxel), topoisomerase-I-inhibitors (Irinotecan and Topotecan),

thymidilate synthetase-inhibitors (Ralitrexed) and antimetabolites like

Gemcitabin have been developed over the past ten years. These drugs interfere with

the rapid cell proliferation. However, cell proliferation is also found in the healthy body

(in the spinal cord, the hair follicles and during pregnancy). This explains the severe

side effect caused by this conventional tumour drugs.

It is believed that the vast majority of tumour cells have to be killed to achieve

complete remission of the tumour. Therefore, the application of high doses is

necessary which aggravates the severe side effects.

One important goal to be achieved in tumour therapy is therefore the development of

anticancer drugs, which selectively accumulate in the tumour, while sparing normal

tissues and organs. This targeted drug delivery lead to a minimisation of the severe

unwanted side effects during cancer therapy.

The monoclonal antibody Herceptin® (Roche) is used for treatment of breast cancer.

The protein kinase inhibitor Glivec® (Novartis) approved by the FDA has shown very

promising results in the treatment of chronic myelogenous leukemia (CML). These

are two examples of new drugs which come closer to the aim of acting selectively on

malignant cells.

1.1.1.2 Selective tumour therapy One of the most promising approaches to improve the selectivity of the tumour

therapy is to deliver active compounds to the tumour site by targeting specific

markers which are only present on or near malignant cells. This concept of

selectively targeting single organs or cell populations with therapeutic “magic bullets”

like antibodies, was first formulated by Ehrlich at the end of the 19th century. Since

2

the development of hybridoma technology for the production of monoclonal

antibodies and with more recent technologies like phage display and humanisation of

antibodies (antibody engineering), Ehrlich’s concept has become more realistic and is

often referred to especially in cancer research.

Today, tumour targeting mainly works on the basis of antibodies or antibody

fragments directed against epitopes only present in malignant tissues.

In general, good targeting crucially depends on two parameters: the quality of the

molecular target (e.g. tumour marker) and the quality of the targeting agent (e.g., a

ligand specific to the target). A good marker should be selectively expressed in the

targeted tissue, it should be abundant and easily accessible.

As different tumour types differ in the pattern of antigens presented on their cell

surfaces, it is very difficult to find an epitope present in many different types of

tumours.

1.1.1.3 Targeting of neovasculature Angiogenesis is defined as the growth of new blood vessels from pre-existing

vessels. It is essential for the development of the vascular network during

embryogenesis. Once the network is in place in the adult, it remains quiescent

outside of certain physiological processes such as wound repair, inflammation and

the female menstrual cycle. Angiogenesis is a fundamental process for the

development of various diseases like inflammation, diabetic retinopathy, rheumathoid

arthritis and cancer. [Birchler et al. 1999; Bischoff, 1997].

Much of our current understanding of angiogenesis stems from studies on tumoural

angiogenesis, as tumour growth strongly depends on neovascularisation. The

amount of newly grown blood vessels growth can correlate with a poor prognosis in

several tumour types [Rosen, 2000]. Each tumour arises from a single cell that has

been transformed by one or more events. This transformed cell can form small

clones, initially coopting normal host vessels, growing only to several millimetres in

size before their supply of nutrients become limited. At this point, the tumour may lie

dormant for prolonged periods until ultimately undergoing destruction by immune

3

system or switching to an angiogenic phenotype which can evolve into a clinically

relevant tumour, invade and metastasise.

Because angiogenesis occurs only rarely in healthy adults, anti-angiogenesis therapy

is likely to avoid some of the cytotoxicity associated with conventional chemotherapy

modalities. Targeting markers of angiogenesis offers several theoretical advantages

over targeting tumour markers directly [Denekamp, 1990; Burrows and Thorpe,

1994b, Ran et al., 1998]. First antibodies against markers of angiogenesis can be

used for different tumour types, as neovascularization appears to be a general

feature of tumour growth and invasiveness. A single reagent specific for

angiogenesis could be used for diagnosis or therapy of different kinds of tumours, as

they rely on similar types of blood vessels and might have similar upregulated

endothelial markers [Viti et al., 1999; Bloemendal et al. 1999]. Second, local

interruption of tumour vasculature will result in tumour cell death of great extense,

since thousands of tumour cells rely on one capillary for oxygen and nutrient supply.

Third, non-tumour cells are unlikely to acquire mutations that render them resistant to

therapy.

Markers of angiogenesis have to be specific, accessible and abundant if they have to

serve as a target for therapeutic or diagnostic intervention. To date, only few good

quality markers of angiogenesis located either on endothelial cells or in the modified

extracellular matrix are known. Most existing candidate markers are also expressed

in some normal tissues, thus limiting their usefulness. Systematic ex vivo

investigations of tumour endothelial structures using proteomic techniques [Schnitzer

et al., 1998; Schnitzer et al., 1995], biopanning of phage display libraries [Rouslathi,

2000; Kolonini et al., 2001; Hogenboom et al., 1999; Rousch et al. 1998; Zardi et al.,

1987; Carnemolla et al., 1989; Castellani et al., 1994] or transcriptomic techniques,

such as serial analysis of gene expression [Croix et al., 2000], are revealing new

candidate tumour endothelial markers.

Established markers of angiogenesis located on the cell surface of tumour

endothelial cells are the integrins αvβ3 and αvβ5 [Sipkins et al., 1998; Arap et al.,

1998], endoglin [Burrows et al., 1995], vascular endothelial growth factor (VEGF) and

VEGF-receptor complex [Brekken et al., 1998], prostate specific membrane antigen

4

(PSMA) [Chang et al., 1999], aminopeptidase N [Pasqualini et al., 2000], CD62E

[Langley et al., 1999] and also phosphatidylserine phospholipids [Ran et al., 2002].

Other markers of angiogenesis are part of the modified ECM. Probably one of the

most selective markers of angiogenesis, the extra domain B of fibronectin (ED-B), is

located in the modified ECM within the tumour environment [Zardi et al., 1987;

Carnemolla et al., 1989]. With some very rare exceptions (uterus, ovaries) ED-B is

undetectable in normal tissues, but exhibits a much greater expression in foetal and

tumour tissues as well as during wound healing [Castellani et al., 1994]. Another

interesting marker of the tumour-associated ECM is the large tenascin-C isoform

[Borsi et al., 1996].

1.2 Discovery of novel accessible tissue-specific targets To serve as target in clinical diagnosis and/or therapy, a tissue-specific marker has

not only to be specific and abundant, but should ideally be readily accessible from

the blood stream for intravenously administered drugs. Thus, at least in theory,

molecules on the luminal plasma membrane of endothelial cells are likely to be ideal

targets, if they are expressed tissue-specifically and abundantly enough.

As mentioned before, angiogenesis performs a key function in the development of an

invasive, metastatic tumour. Therefore, the closer investigation of the behaviour of

endothelial cells and the study of angiogenesis may lead to the discovery of novel

accessible tumour markers.

For these reasons a variety of research models systems (in vivo, ex vivo and in vitro)

were established.

Even though there are other molecular classes which could in principle represent

interesting targets for biomolecular intervention (e.g., phospholipids,

polysaccharides), a main focus in target discovery is the analysis of proteins. This is

due in part to the wide varieties of patterns of expression that proteins may display in

different tissues, and in part to the fact that powerful technologies are avaible for the

detection of proteins (“proteomics”). Proteome analysis/proteomics means the

5

analysis of proteins expressed by an organism or a specific tissue. During disease, a

change of the proteome can occur.

The following sections outline the different models for the discovery of vascular

targets and the various proteomic techniques for the analysis of membrane proteins.

1.2.1 In vitro model systems for the discovery of novel markers of neovasculature

In spite of many differences which can be observed between different types of

cancer, some common features are characteristic for aggressive, rapidly growing

solid tumours. In addition to features such as the genomic instability of tumour cells

and their deranged proliferation and attachment behavior, the tumour environment is

also characterized by the presence of new blood vessels and (in spite of them) by the

insufficient blood perfusion of the tumour mass, leading to hypoxia, serum starvation

and pH changes.

Our group and others have tried to mimic the tumour environment in vitro, using cell

cultures (primary endothelial cells, primary cultures of fibroblasts, etc.) and studying

the changes in gene expression as a response to environmental changes (pH, serum

starvation, hypoxia, etc.). In our experience, the combined use of transcriptomic

analysis (using the Affymetrix gene chip system) and proteomic investigations (by

2D-PAGE and mass spectrometry) is often beneficial, as the two technologies are

often complementary in identifying candidate genes whose patterns of expression

are regulated by environmental changes [Scheurer et al., 2004].

The study of gene expression in endothelial cells isolated from tumours and normal

tissues may allow a closer analysis at the patterns of gene expression in tumour

vascular structures. The quality of endothelial cell purification is crucial for these

types of studies, both in terms of separation from other cell types and in terms of

speed, preventing post-separation changes in the abundance of mRNAs and

proteins. Most studies performed so far have studied levels of gene expression using

transcriptomic methods, such as cDNA subtractive hybridization procedures [Wyder

6

et al.,2000], serial analysis of gene expression (SAGE) [Carson-Walter et al., 2001]

and gene chip-based methods. A group at Genentech has crossed gene expression

data obtained comparing colorectal cancer and normal mucosa with a database of

genes known to be expressed in endothelial cells, thus identifying putative markers

with differential expression in tumour endothelial cells compared to endothelial cells

in normal tissues [Kahn et al., 2000; Fujiwara et al., 2000; Gerritson et al., 2002].

Interestingly, the most attractive candidate resulting from this study (stanniocalcin) is

also one of the most strongly up-regulated genes in in vitro model experiments, when

shifting cell culture pH to an acidic value [Bumke et al., 2003].

In principle, proteomic investigations should be possible if sufficient amounts of

endothelial cells and the associated ECM components can be recovered from

tumours, either by cell purification [Alessandri et al.; 1999] or by laser capture

microdissection [Craven et al., 2002].

The experimental approaches described above can, at best, suggest candidate

markers of angiogenesis. An experimental confirmation, however, requires the

generation of specific monoclonal antibodies and an extensive immunohistochemical

analysis of expression patterns in normal and pathological specimens.

1.2.2 In vivo and ex vivo model systems for the identification of vascular targets

In principle, the most direct way to assess differences in protein abundance between

the tumour endothelium and the normal endothelium would consist in the in vivo

labeling of vascular structures, followed by rapid recovery and comparative proteomic

analysis of the proteins in the two samples.

The group of Jan Schnitzer has pioneered the use of colloidal silica for the in vivo

coating of vascular structures in tumours and in normal organs [Jacobson et al.,

1992; Czarny et al., 2003; Oh et al., 2004]. This physical modification allows the

recovery (by centrifugation and fractionation) of silica-coated structures (luminal cell

plasma membranes and caveolae of the endothelium), providing ideal material for

7

proteomic investigations, for example by immunization [McIntosh et al., 2002] or by

2D-PAGE.

De la Fuente et al. have described the ex vivo perfusion of lungs isolated from normal

and hyperoxic rats with Sulfo-NHS-LC-biotin [De La Fuente et al., 1997]. After SDS-

PAGE, the biotinylated proteins were visualized using a chemiluminescence

substrate for the streptavidin-horseradish peroxidase conjugate, outlining differences

in rats exposed to hyperoxia for 48-60 hours. The authors claim that this approach

could provide a starting point for the identification of novel, specific proteins relevant

to the response to lung injury.

1.2.3 Proteomic technologies for target identification Membran proteins and ECM proteins represent the most attractive classes of

proteins for ligand based targeting application. Proteomic technologies are often at

their technical limits for the characterisation of these proteins, because of low

abundance and/or limited solubility.

Membrane proteins are either situated at the interface between the cell and the

surrounding environment or at the interface of subcellular compartments, and

perform key functions such as cell-to-cell recognition and transport of ions and

solutes, as well as acting as receptors for forwarding the diverse signals that reach

the cell or subcellular compartment. Membrane proteins are often important for a

cell’s survival, and they are often suitable targets for pharmaceutical intervention.

Indeed, more than two-thirds of the known protein targets for drugs are membrane

proteins [Stevens et Arkin, 2000].

The expression of membrane proteins can vary as a consequence of disease,

potentially providing targets for the selective delivery of pharmaceuticals.

The investigation and quantitation of membrane proteins remains a challenging task

for several reasons. First, membrane proteins are typically low abundant proteins.

The dynamic range in protein abundance (copy numbers per cells or tissues) is

believed to cover up to nine orders of magnitude, and thus the analysis of low

abundant proteins is very difficult using any technique [Rabilloud, 2002]. Second,

8

membrane proteins are hydrophobic proteins designed to be soluble in lipid bilayers

and consequently are difficult to solubilize in aqueous media [Santoni et al., 2000].

While these techniques are compatible with the limiting requirements for membrane

proteins, they are nevertheless suitable for the analysis of normal, soluble proteins,

which could also be possible targets if specific, accessible and abundant enough.

The following chapters outline some methods, which have been applied for the

investigation of membrane proteins.

1.2.3.1 Gel-based proteomic technologies 1.2.3.1.1 Two-dimensional polyacrylamide gel electrophoresis Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a powerful

method for the analysis of complex protein mixtures extracted from cells, tissues or

other biological samples. This technique, which was first introduced by P.H. O’Farrell

[Farrel, 1975] and J. Klose, [Klose, 1975] sorts proteins according to two independent

properties in two discrete steps: the first dimension step, isoelectric focusing (IEF),

separates proteins according to their isoelectric points (pI); the second-dimension

step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins

according to their molecular weights (MW). 2D-PAGE is a core technique in

proteome analysis, which usually includes sample preparation, 2D-PAGE, post

separation image analysis of the stained gel and protein characterization by mass

spectrometry. Microanalytical techniques were developed, which allowed the

identification of proteins at the amounts available from 2D-PAGE. Data about entire

genomes (or substantial fractions thereof) are available for a number of organisms,

allowing rapid identification of the gene encoding a protein separated by 2D-PAGE.

However, despite all the advantages of the 2D-PAGE technique, there are still

limitations, which are linked to the chemical diversity of proteins in a cell or tissue and

to their different abundance. These limitations are the reasons for not submitting our

complex biotinylated protein extracts to 2D-PAGE.

9

1.) The comparison between the number of actin molecules (ca. 108 molecules

per cell) and the number of some cellular receptors (ca. 100 – 1000 molecules

per cell) present in a cell, reveals a dynamic range of up to 1´000´000 between

the most abundant and the least abundant proteins [Rabilloud, 2000]. 2D-

PAGE in contrast can only display differences in protein concentration in the

range of 100-10´000. Since membrane proteins are typically little abundant,

they are often underrepresented on 2D-PAGE gels.

2.) A complex protein mixtures lead to comigrating proteins which compromise

quantitative analysis based on the assumption, that one protein is present per

spot [Gygi et al., 2000].

3.) The membrane proteins as most promising target structures tend to precipitate

during IEF, when they concentrate at their pI.

4.) The strong anionic detergent SDS, which is known to solubilize almost any

protein, interferes with the isoelectric focusing step in the first dimension of

2D-PAGE and can only be used in a concentration below 0.25% [Ames et

Nikaido, 1976; Harder et al., 1999]. Therefore, our biotinylated protein extract

solubilized in lysis buffer containing 2 % SDS could not be submitted to 2D-

PAGE analysis.

As a consequence, alternative approaches rely on one-dimensional SDS gel

electrophoresis (1D-SDS PAGE) for the separation of membrane proteins, replacing

the IEF by another separation technique in the first dimension.

1.2.3.1.2 Combination of chromatography and 1D-SDS-PAGE The solubility problem of membrane proteins during the IEF step in 2D-PAGE

stimulates the search for alternative separation methods, orthogonal to SDS-PAGE.

Complex protein mixtures can be separated according to several parameters,

including the retention on a chromatographic column and/or the molecular weight.

The separation of individual fractions after chromatography by means of 1D-SDS-

PAGE generates two- or multi-dimensional patterns and facilitates the resolution of

different proteins.

10

A wide variety of high-performance liquid chromatography (HPLC) systems has been

employed originally for the purification of membrane proteins [Thomas et McNamee,

1990; Welling et al., 1987]. These include size-exclusion-HPLC, ion-exchange-HPLC,

bioaffinity chromatography, reversed-phase-HPLC (RP-HPLC) and hydroxyapatite-

HPLC (HAP-HPLC) with SDS. Since those chromatographic methods allow the

separation of contaminants from membrane proteins, the question arises, whether

one can also part different membrane proteins contained in a membrane extract

applying chromatography in the first dimension.

Horigome and colleagues combined ceramic HAP-HPLC with 1D-SDS-PAGE for the

investigation of membrane proteins isolated from rat erythrocyte membranes and rat

liver microsomes [Horigome et al., 1989]. The HAP-HPLC was performed with a

buffer system, which contained 1% of SDS and sodium phosphate up to a

concentration of 0.5 M. In another study, membrane proteins from rat liver rough

microsomes were efficiently resolved with a protein recovery of more than 90% by

HAP-HPLC, using 1% sodium cholate as detergent [Ichimura et al., 1995].

Hydroxyapatite chromatography was introduced in 1956 by Tiselius [Tiselius et

Hjerten, 1956]. In HAP-HPLC, biomolecules are separated according to their different

interactions with hydroxyapatite, whose molecular formula is Ca10(PO4)6(OH)2.

Positively charged ammonium groups (e.g., side chains of lysine residues) are

attracted by the phosphate groups on the column and repelled by the calcium ions;

the situation is the opposite for carboxylic acids [Gorbunoff, 1984a; Gorbunoff,1984b;

Gorbunoff et Timosdheff, 1984 ].

HAP-HPLC allows the use of strong detergents, which is very advantageous when

working with membrane proteins. However, the same proteins are often found in

more than one fraction, thus hampering the overall resolution of the two-dimensional

separation process.

Using SDS-PAGE for the one-dimensional separation of membrane proteins and

micro capillary liquid chromatography-electrospray ionization tandem mass-

spectrometry (µLC-ESI-MS/MS) for the analysis of peptides generated by digesting

the protein migrating to a particular zone of the gel, Simpson and co-workers

identified 284 proteins, including 92 membrane proteins [Simpson et al., 2000].

Although this method is suitable for cataloguing proteins contained in membrane

11

fractions, it is inherently not quantitative and therefore not suitable for the detection of

differences in the membrane-protein profile of cells representing different states.

More in general, the combination of (i) fractionation of sub-cellular organelles (ii)

chromatography in the presence of SDS and (iii) 1D-SDS-PAGE appears to be a

robust avenue for the comparison of relative protein abundance in different

cells/tissues.

1.2.3.2 Gel-free proteomic technologies 1.2.3.2.1 Mass spectrometry-based methods Even when protein bands (or spots) are well resolved in polyacrylamide gel

electrophoresis, the routine use of this technique for the comparison of the relative

abundance of proteins in different samples is limited to the detection of big changes

(e.g., > 3-fold change in relative abundance). Special techniques (such as

biosynthetic or post-separation isotope labeling, 2D-difference fluorescence gel

electrophoresis) are not always compatible with the requirements for the discovery of

novel markers of angiogenesis (e.g., in vivo protein biotinylation), but have been

used with success for the studies of cells cultured in different conditions [Aebersold

and Mann, 2003]. Furthermore, methodologies such as 2D-PAGE are labor intensive

and do not lend themselves easily to the analysis and comparison of several dozens

of samples.

The continuous advances in the field of biological mass spectrometry has now made

it possible to routinely identify proteins from the corresponding endoproteolytic

peptides, at total protein amounts in the femtomole range. The combined use of

multidimensional liquid chromatography (LC) and tandem mass spectrometry

(MS/MS) has made it possible to identify hundreds of proteins in the same sample

containing tryptic peptides [Link et al., 1999]. In a large-scale analysis of the yeast

proteome, more than 100 membrane proteins were identified [Washburn et al., 2001].

More recently, a modified methodology has been developed for the application of

mass spectrometry to the study of membrane proteins [Wu et al., 2003]. A

12

combination of membrane sheets enrichment at high pH, followed by Proteinase K

treatment and LC/MS analysis, has allowed the identification of > 400 of membrane

proteins in a rat brain homogenate. All these methodologies, however, do not yield

information about relative protein quantity at present, and cannot be used for the

comparison of membrane protein abundance in different complex specimens.

In 1999, Aebersold and coworkers introduced the concept of isotope-coded affinity

tags (“ICAT”) for the stable non-radioactive isotopic labeling of proteins, compatible

with protein identification and relative quantification in different biological specimens

[Gygi et al., 1999]. In its original implementation, the ICAT technology consists in the

biotinylation of cysteine residues in proteins with reactive derivatives of biotin,

carrying a linker arm with hydrogen or deuterium atoms. These “light” and “heavy”

biotin derivatives serve a dual purpose. First, they allow to reduce the complexity of

tryptic peptides to be analyzed in a gel-free mass-spectrometry experiments (they

can be purified on affinity resins; only few tryptic peptides in a protein contain a

cysteine residue). Second, the labeling of peptides from two different samples with a

light or heavy tag allows the use of LC-MS/MS methodologies for the relative

comparison of protein abundance in the two samples. The relative protein abundance

is in fact reflected in the relative intensity of the mass spectrometry signals of the

corresponding biotinylated peptides, which are separated in the m/z axis by the

number of Daltons corresponding to the number of atoms which are either hydrogen

or deuterium in the biotin derivative tag.

It is almost certain that modern mass spectrometric procedures and instrumentation

(such as the use of Fourier transform ion cyclotron (FT-ICR) and MALDI-TOF time of

flight (MALDI-TOF-TOF) spectrometers [103]), which offer unprecedented resolution

and sensitivity, will contribute to the increased use of mass spectrometry-based

methods for gel-free proteomic analysis. However, the study of membrane proteins

will also require improved methodologies for the chemical modification and recovery

of peptides from these low abundant, hydrophobic proteins.

13

1.2.3.2.2 Ligand-based methods Traditionally, the first markers of angiogenesis have been discovered either by limited

proteolysis of purified protein preparations [Zardi et al., 1987], or by animal

immunization with biological samples derived from tumours, followed by an extensive

immunohistochemical analysis of the resulting hybridomas [Liu et al., 1997; Chang et

al., 1999].

The introduction of recombinant antibody technologies, and in particular of antibody

phage technology [Winter et al., 1994], has greatly facilitated the production of good-

quality monoclonal antibodies without immunization. These technologies are

particularly efficient when pure antigen preparations are available [Viti et al., 2000],

but antibodies have also been generated from phage libraries against “difficult”

antigens [Hogenboom et al., 1999].

It is difficult to imagine that antibody-based chips may facilitate the study of the

relative abundance of membrane proteins in different biological specimens [Elia et

al., 2002]. Nonetheless, if larger public-domain collections of monoclonal antibodies

become available in the future, it should be possible to use fluorescence-activated

cell sorters (FACS) and/or immunohistochemistry with tissue arrays [Schraml et al.,

1999] for the relative quantitation of membrane proteins in different cells/tissues.

Ruoslahti, Pasqualini and co-workers have pioneered the in vivo biopanning of

peptide phage libraries, in an attempt to identify binding specificities against different

vascular addresses in different tissues and/or tumours [Pasqualini and Rouslathi,

1996; Rajotte et al., 1998]. Among others, peptides specific to integrins and to CD13

were identified with this procedure. However, the real potential of this technology

remains to be demonstrated, considering that the use of peptides on tissue sections

is often less efficient than the use of antibodies (which normally display a higher

affinity for the antigen), and in the absence of quantitative biodistribution studies and

clinical studies with purified preparations of the vascular-targeting peptides.

14

1.2.3.3 Cell surface biotinylation Both the gel-based proteomic analysis as well as the mass spectrometry based

techniques suffer from the high complexity of the examined sample. Cell surface

biotinylation leads to a reduction of complexity by capturing only the labelled proteins

on Streptavidin sepharose.

Cell surface biotinylation is based on the chemical modification of certain amino

acids. For practical application only primary amino groups or thiol groups in proteins

can be used for chemical modification strategies.

Depending on the sample, one or more targets can be linked with a chemical

reagent, which consists of a binding part to the protein, a linker and a group that

permits the selective capturing of the modified proteins [Lottspeich and Zorbas,

1998].

The selective labelling of the cell surface proteins with a cleavable biotin label has

been previously described by the group of G. Busch [Busch et al., 1989]. Busch and

co-workers biotinylated human erythrocytes and rat hepatocytes. They purified the

biotinylated proteins after cell lysis on avidin-agarose.

De la Fuente et al., as mentioned above, perfused lungs from rats with Sulfo-NHS-

LC-biotin. [De la Fuente et al., 1997]. The spectrum of proteins biotinylated via the

vasculature was distinct from that of the biotinylated lung homogenate.

A. Braendli [Braendli et al., 1990] isolated biotinylated proteins with streptavidin-

agarose. The biotinylation reagents used in this work were Sulfo-NHS-SS-biotin (see

Fig. 1.2 ) and Sulfo-NHS-LC-biotin (see Fig 1.3).

1. Sulfo-NHS-SS-Biotin consists of four parts:

• Sulfo-NHS-SS-biotin: The charged sulfate-group confers the water

solubility and limits the penetration through the membrane.

15

• Sulfo-NHS-SS-biotin: The N-hydroxysuccinimide ester reacts to form an

amide bond with primary amino groups. (see Fig. 1.1 )

Fig. 1.1 The reaction of Sulfo-NHS-Biotin with primary amines of proteins.

• Sulfo-NHS-SS-biotin: The linker contains a disulfide bond, which can be

cleaved by reducing agents. This is important for the release of

biotinylated proteins from streptavidin or avidin.

• Sulfo-NHS-SS-biotin: To purify the labeled proteins, biotin is bound on

streptavidin and proteins without biotin tag are washed away.

Fig. 1.2 Molecular structure of Sulfo-NHS-SS-biotin

16

2. Sulfo-NHS-LC-biotin consists also of four parts which were almost identical as

the four parts of the Sulfo-NHS-SS-biotin, except the cleavable SS-bond of the

above described reagent is replaced by an uncleavable, hydrophobic linker.

That explains the harsh conditions, which are to be used for the elution of the

biotinylated proteins from streptavidin or avidin.

Fig. 1.3 Molecular structure of Sulfo-NHS-LC-biotin

17

1.3 In vivo biotinylation technology Driven by the need for more selective drugs discussed above and inspired by the

work of De La Fuente and co-workers (De La Fuente et al., 1997), who performed ex

vivo perfusion of isolated rat lungs with a biotinylation reagent (see above), Rybak et

al. (2004) have set up in our laboratory a novel technology – termed “in vivo

biotinylation” – for the discovery of tissue-specific targets accessible from circulation

(see Fig. 1.4).

Target tissue (e.g., tumor or

diseased organ)

Blood vessel

Biotin BiotinBiotinBiotin Biotin

Perfusion

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Purification of biotinylated proteins on streptavidin

Comparative proteomic analysis

Targetidentification

Biotinylation ofaccessible proteins

Fig. 1.4: Strategy of the in vivo biotinylation technology for the

discovery of accessible tissue-specific targets for selective drug delivery. (See text for explanation.)

18

This approach is based on the terminal perfusion of rodents with a charged, activated

ester derivative of biotin, which cannot diffuse through biological membranes.

Proteins, glycolipids and phospholipids carrying accessible primary amino groups

may be labeled in this process. Biotinylated proteins can efficiently be purified on

streptavidin resins and submitted to a comparative proteomic analysis, which may

reveal candidate targets which are differentially expressed in organs and diseased

tissues (e.g., tumours).

1.4 Experimental overview and aims of this diploma thesis Rybak et al. had already routinely peformed in vivo biotinylation experiments with

tumour-bearing mice according to the scheme in Fig. 1.5, which also represents the

main experimental workflow of the diploma thesis presented here: F9

teratocarcinoma cells were cultured and injected subcutaneously in SvEv/129 mice

inducing the growth of an allograft tumour. The mice were subjected to a perfusion

procedure comprising in vivo biotinylation. Subsequently, biotinylation of vascular

structures was proven by histochemical staining of organ and tumour sections with a

streptavidin conjugate. After homogenization of tissue specimens, proteins were

extracted and protein concentration was determined in each sample. These protein

extracts were submitted either to a streptavidin blot analysis to check the biotinylation

of proteins or to a streptavidin affinity purification followed by a comparative

proteomic analysis of the biotinylated proteins by two different approaches (a gel-

based analysis based on SDS-PAGE and tryptic in-gel digestion of resulting protein

bands or a gel-free “shot-gun” analysis based on the tryptic digestion of the whole

mixture of captured proteins directly on the resin), which both comprised finally the

identification of biotinylated and thus directly accessible proteins by mass

spectrometry analysis of the corresponding peptides.

The aim of this diploma thesis was to improve several suboptimal aspects of this

technique.

19

protprot

Culture+ injection of F9 cellsto induce tumours in

mice

Perfusion of tumor-bearing mice with

biotinylation reagent

Histochemistry forthe detection of

biotinylatedstructures

Organ homogenisation

SA blot to check biotinylation of

eins

Protein extraction

Protein determination

Gel-basedproteomicanalysis

Gel-freeproteomicanalysis

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Culture+ injection of F9 cellsto induce tumours in

mice

Perfusion of tumor-bearing mice with

biotinylation reagent

Histochemistry forthe detection of

biotinylatedstructures

Organ homogenisation

SA blot to check biotinylation of

eins

Protein extraction

Protein determination

Gel-basedproteomicanalysis

Gel-freeproteomicanalysis

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

1.4.4Alternative perfusion reagents

1.4.3 Optimisation of the gel-free proteomic analysis

1.4.2 Optimisation of the gel-based proteomic analysis

1.4.1 Optimisation of the tumour perfusion

Fig 1.5 Workflow of the in vivo biotinylation and subsequent analyses. The red arrows indicates the steps, which were aimed to be optimized in the diploma thesis presented here. Numbers refer to the following subchapters describing the optimisation approaches.

20

1.4.1 Optimisation of the tumour perfusion The previous perfusion procedure was performed according to the following workflow

shown in Fig. 1.6:

Surgery

Washing step with PBS

Biotinylation step withSulfo-NHS-LC-biotin in

PBS

Quenching step withTris in PBS

Excision of organs

PERFUSION

Omission of thewashing step

Addition of dextran to the

perfusion solutions

Perfusion with a higher flow rate

Warming of the animal and the

perfusion solutions

Surgery

Washing step with PBS

Biotinylation step withSulfo-NHS-LC-biotin in

PBS

Quenching step withTris in PBS

Excision of organs

PERFUSION

Omission of thewashing step

Addition of dextran to the

perfusion solutions

Perfusion with a higher flow rate

Warming of the animal and the

perfusion solutions

Fig 1.6 Workflow of the tumour perfusion procedure. The red arrows indicate the optimization attempts.

F9-tumour bearing mice were perfused with a flow of ~1.5 ml/min. Blood and cells

were washed away with PBS, the animal was perfused with Sulfo-NHS-LC-biotin,

and excessive biotinylation reagent was quenched with 50 mM Tris in PBS.

Applying this procedure, normal organs could be biotinylated successfully according

to the histochemical and proteomic analysis. However, in most cases the tumour was

biotinylated not at all or only a small part, probably due to incomplete or lacking

perfusion of this type of tissue.

21

Therefore, several optimisation approaches were tested aiming at the improvement

of the tumour biotinylation:

1) In the original protocol applied so far, the perfusion was performed with a flow

rate of about 1.5 ml/min. However, the heart minute volume of a mouse, that

means the rate with which the heart pumps the blood through the circulation in

the living animal, is 11–36 ml/min (!). Thus, it was hypothesized that the

incomplete perfusion of the tumours could be due to a too low perfusion flow

rate. An optimisation approach featuring higher perfusion flow rates was

attempted.

2) In vivo, in spite of the high interstitial pressure and the irregular vasculature

blood can perfuse solid tumours, otherwise the tumour cells would be lacking

oxygen and nutrient supply. To investigate, why the tumours are obviously

perfused in vivo, but difficult to access in the artificial perfusion procedure, we

compared the accessibility of vascular structures for Bisbenzimide H33342

(Hoechst dye), a nuclei-staining fluorescent dye, either by intravenous

injection or by perfusion.

This experiment gave evidence that the PBS washing steps prior to perfusion

with the active reagent might reduce the accessibility of the vessels for the

respective component.

These findings suggested the omission of the washing step prior to the

perfusion with biotinylation reagent.

3) The inefficient perfusion results in solid tumours might also be a result of a

collapse of microvessels, due to a too low colloidal oncotic pressure of the

perfusion solutions, which could lead to a compression of the microvessels.

Thus, in vivo biotinylation experiments were performed as before but omitting

the washing step prior to the perfusion with the biotinylation reagent, and with

the perfusion solutions containing 10 % (w/v) Dextran 40, a substance which

binds water molecules and acts as plasma expander increasing the oncogenic

pressure.

4) The reduced body temperature of the mouse in anesthesia and during the

perfusion might lead to a circulatory shock of the mouse. In a physiological,

unconscious self-protection mechanism, the cool environment as well as the

injury during surgery possibly leads to a vasoconstriction in the periphery of

22

the body aiming at the maintenance of the function of the more important inner

organs. By warming the the perfusion solutions to the body temperature of the

mouse (~38°C) and keeping the animal warm in anesthesia as well as during

surgery and perfusion, we aimed at the better perfusion of tumours.

1.4.2 Optimisation of the gel-based proteomic analysis The previous gel-based proteomic analysis was performed according to the following

workflow presented in Fig. 1.7:

Peptide analysis by µLC-MS/MS and

protein identification0

20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00

Capture on streptavidinsepharose

SDS-PAGE and comparison of protein

band pattern

Tissue protein extractfrom in vivo biotinylated

mouse

Elution fromstreptavidinsepharose

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Tryptic in-gel digestion

Excision of protein bands



20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00



band pattern


mouse


Biotin

Biotin

wash

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin



Protein concentrationOn the resin

Sample concentrationby spin dialysis

Biotin

Biotin

Biotin

Biotin

Biotin

BiotinB

iot in

Bio ti n



20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00



band pattern


mouse


Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin





20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00



band pattern


mouse


Biotin

Biotin

wash

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin



Protein concentrationOn the resin

Sample concentrationby spin dialysis

Biotin

Biotin

Biotin

Biotin

Biotin

BiotinB

iot in

Bio ti n

Biotin

Biotin

Biotin

Biotin

Biotin

BiotinB

iot in

Bio

t in

Bio ti n

Bio ti n

Fig 1.7 Workflow of the gel-based proteomic analysis. The red arrows indicate the steps which were aimed to be optimized.

Tissue protein extracts from in vivo biotinylated, tumour-bearing mice were captured

on and eluted from SA sepharose. After purification the labelled proteins were loaded

23

on a gel, the bands were excised, a tryptic in-gel digestion was performed and the

resulting peptides were analysed by µLC-MS/MS.

Only few proteins were identified using this gel-based method as a consequence of

the low number and intensity of the protein bands.

Therefore, two different optimisation approaches were implemented at different

stages of the workflow aiming at the enhancement of the protein amount loaded on

the gel. The first approach aspired to enhance the protein concentration on the resin.

The second approach attempted to concentrate the sample after purification by spin

dialysis.

1.4.3 Optimisation of the gel-free proteomic analysis The original gel-based proteomic analysis was performed according to the following

workflow presented in Fig. 1.8 :

Tryptic „on-resin“digestion


protein identification («shot-gun»approach)0

20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00


Tissue protein extractfrom in vivobiotinylated

mouse

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin Tryptic „on-resin“digestion


protein identification («shot-gun»approach)0

20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00


Tissue protein extractfrom in vivobiotinylated

mouse

Biotin

Biotin

wash

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

NH2+

NH2+

COO-

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

COO-

NH2+

NH2+

NH2+

COO-

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

COO-

NH2+

NH2+

NH2+

COO-

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

COO-

NH2+

NH2+

NH2+

COO-

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

PO4-

Ca 2+

COO-

NH2+

Fig 1.8 Workflow of the gel-free proteomic analysis. The red arrows indicate the steps which were aimed to be optimized.

24

The entire portion of proteins from the tissue protein extract, which was captured on

SA sepharose was directly digested on the resin by trypsin. The resulting peptides

were submitted to mass spectrometry analysis.

More proteins were identified compared to the gel-based proteomic approach.

However, further optimisation was desired to identify even less abundant proteins.

One possibility to facilitate mass spectrometric analysis is to reduce the sample

complexity. Therefore, within the framework of this diploma thesis as hydroxyapatite

based chromatography method for the pre-fractionation of biotinylated proteins was

set up, which allows the presence of SDS in all purification steps making sure, that

also hydrophobic membrane proteins were included in the analysis.

1.4.4 Alternative perfusion reagents In the gel-free proteomic analysis of biotinylated proteins from in vivo biotinylated

mice, in addition to the expected proteins from cell plasma membrane and

extracellular matrix, a lot of intracellular proteins were identified (see chapter 3.3 and

not shown data from Giuliano Elia). Even though the biotinylation reagent carried a

charged sulfate group, it could nevertheless penetrate biological membranes to a

certain extent. To overcome this problem two further optimisation attempts were

made (see Fig. 1.9):

1) Instead of Sulfo-LC-Biotin, the cleavable, disulfide containing Sulfo-NHS-SS-

Biotin was used as biotinylation reagent.

Peirce and co-workers [Pierce at al., 2004] observed that cell surface

biotinylation of permeabilized cells with Sulfo-NHS-SS-Biotin lead in a

significant proportion to biotinylation of the whole intracellular compartment,

while this was not the case, when Sulfo-NHS-SS-biotin was used as

biotinylation reagent.

This may be a result of cleavage of the disulfide bond due to the reducing

intracellular environment.

2) High molecular weight polymers should not be able to diffuse through

biological membranes. Thus, a dextran-biotin-NHS derivative was

synthesized, providing the following features:

25

a) biotin residues for affinity capturing on streptavidin

b) NHS-groups for the reaction with primary amines

c) a fluorescent label for the direct detection by fluorescent microscopy of

the corresponding organ sections or the direct detection of protein

bands in a fluorescence imager

d) a high molecular weight compund should not be able to penetrate

biological membranes

Perfusion with alternativebiotinylation reagents:

•Sulfo-NHS-SS-Biotin

•Dextran-biotin-NHS derivative

Synthesis of the Dextran-biotin-NHS-derivative

Perfusion with alternativebiotinylation reagents:

•Sulfo-NHS-SS-Biotin

•Dextran-biotin-NHS derivative

Synthesis of the Dextran-biotin-NHS-derivative

Fig 1.9 Alternative perfusion reagents for the optimization of the in vivo biotinylation technology

26

2 MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemicals and reagents

Acepromazin (Prequillan) FATRO S.p.A. (Ozzano Emilia,

BO, USA)

Accustain Hematoxilin solution Gill Nr.2 #GHS-2-16, Sigma (St. Louis,

MO, USA)

Acetic Acid #45731, Fluka (St. Louis, MO,

USA)

Acetone, multisolvent #AC0310, Scharlau (Barcelona,

Spain)

Acetonitrile (HPLC grade) #AC0329, Scharlau (Barcelona,

Spain)

Ammonium hydrogen carbonate (NH4HCO3) #09830, Fluka (St. Louis, MO,

USA)

Antibiotic-Antimycotic #15240-062, Gibco (Invitrogen

cooperation, Carlsbad, CA,

USA)

Aprotinin #A-6279, Sigma (St. Louis, MO,

USA)

BCA Protein Assay Reagent Kit #23227, Pierce (Rockford, IL,

USA)

Bovine serum albumin #A-7030, Sigma (St. Louis, MO,

USA)

Bromophenolblue #161-0404, BioRad (Hercules,

CA, USA)

Calcium chloride (CaCl2 x 2 H20) #2382, Merck (Darmstadt,

Germany)

Complete EDTA free (proteinase inhibitor cocktail) #1873580, Roche (Basel,

Switzerland)

27

Crosslinker: DST Disuccinimidyl Tartrate BC-05 #20589, Pierce (Rockford, IL,

USA)

Cryo-embedding compound for medium and lower

temperature #350100, Microm (Walldorf,

Germany)

Cryo-embedding compound for higher and medium

temperature #358100, Microm (Walldorf,

Germany)

Cytochrome C from bovine heart #C-2037, Sigma (St.Louis, MO,

USA)

D-Biotin #12115, usb (Cleveland, OH,

USA)

Developer Adefo T-Matic #00034, Alexander Pidt GmBH

(Herten, Germany)

Dextran 40 #17-0270-01

AmershamBiosciences

(Buckinghamshire, GB)

Dextran (modified), biotin, 70’000 MW, lysine fixable

(BDA-70’000) #D-1957, Molecular Probes

(Eugene, OR, USA)

Diethyl ether #Et0080, Scharlau (Barcelona,

Spain)

Dimethylsulfoxide #D2650, Sigma (St. Louis, MO,

USA)

Dimethylsulfoxide #42642, Fluka (St.Louis, MO,

USA)

N,N-Dimethylformamid #40250, Fluka (St. Louis, MO,

USA)

DTE #H-8255, Sigma (St.Louis, MO,

USA)

DMEM (+Glucose, +Glutamin, -Pyruvate) #41965-039, Gibco (Invitrogen


USA)

28

ECL+plus western blotting detection system #RPN2132, Amersham

Biosciences (Buckinghamshire,

GB)

EDTA #E-1644, Sigma (St. Louis, MO,

USA)

Ethanol #02860, Fluka (St. Louis, MO,

USA)

Fast-Red Powder #F8764-16, Fast Red TR,

Sigma (St. Louis, MO, USA)

Fibrinogen from bovine plasma #F8630, Sigma (St. Louis, MO,

USA)

Filter paper #3030-861, Whatman

(Brentford, GB)

Fluoresceinamin #46930, Fluka (St. Louis, MO,

USA)

Fluorescein sodium salt #F6377, Sigma (St.Louis, MO,

USA)

FP 30/0,2 CA-S Disposable Filter Units Red rim

(0.2µm, sterile, not pyrogen) #10 462 200, Schleicher und

Schuell (Dassel, Germany)

Fixer Adefo T-Matic #00091, Alexander Pidt GmBH

(Herten, Germany)

Fluorescein-5-isothiocyanate #F-143, Molecular Probes

(Eugene, OR, USA)

Foetal bovine serum (FCS) #10106-169, Gibco (Invitrogen


USA)

Formic acid #06440, Fluka (St. Louis, MO,

USA)

Gelatine 2% solution (Typ B:From bovine skin) #9000-70-8, Sigma (St.Louis,

MO, USA)

Glycergel mounting medium #C0563, DAKO corporation

(Carpinteria, CA, USA)

29

Glycerol #17-1325-01, plusone

AmershamBiosciences


Glutathione (GSSG) #G-2140, Sigma (St. Louis,

MO, USA)

Hoechst Dye #B2261, Bisbenzimide H33342

Sigma (St. Louis, MO, USA)

HCl #20-2125-2, Hänseler (Herisau,

Switzerland)

Human serum albumin #A-3782, Sigma (St. Louis, MO,

USA)

Iodoacetamide #I-6125, Sigma (St.Louis, MO,

USA)

Isopentan #59075, Fluka (St. Louis, MO,

USA)

Ketamin (Narketan) Vétoquinol (Belp-Bern,

Switzerland)

Levamisole #L-9756, Sigma (St. Louis, MO,

USA)

Liquid nitrogen

Lysozyme #L-7651, Sigma (St. Louis, MO,

USA)

Magnesium chloride (MgCl2 x 6 H2O) #63065, Fluka (St. Louis, MO,

USA)

β-Mercaptoethanol #M-6250 Sigma (St. Louis, MO,

USA)

Methanol (HPLC grade) #ME0306, Scharlau

(Barcelona, Spain)

Milk powder MIGROS (Zürich, Switzerland)

MilliQ produced by a MilliQ producing

apparatus((RiOs 5 and MilliQ

RG)

Molecular weight marker:

30

• Precision Plus Protein Unstained Standards #161-036, BioRad (Hercules,

CA, USA)

• RPN 800V Full range Rainbow recombinant

protein molecular weight marker #161-0318, BioRad (Hercules,

CA, USA)

MOPS SDS running buffer #NP0001, Invitrogen (Carlsbad,

CA, USA)

Na2HPO4 #6-3300-0, Hänseler (Herisau,

Switzerland)

NaCl #71381, Fluka (St. Louis, MO,

USA)

NaCl 0.9% Ecoflac 500ml #03.730.27, Polymed

(Glattbrugg, Germany)



Naftolol AS-MX Phosphate #N-4875, Sigma (St.Louis, MO,

USA)

NaH2PO4 #6-6104-0, Hänseler (Herisau,

Switzerland)

Nitrocellulose membrane #10401196, Schleicher &

Schuell (Dassel, Germany)

Nonidet P40 Substitute (NP-40) #74385, Fluka (St. Louis, MO,

USA)

Non-Sterile 4mm Millex Syringe Filter Unit (0.2 µm) #SLGVR04NL, Millipore

(Billerica, MA, USA)

Ovalbumin #A-5503, Sigma (St.Louis, MO,

USA)

Pepsin porcine gastric mucosa #P7000, Sigma (St Louis, MO,

USA)

SDS #17-1313-01, plusone

AmershamBiosciences


SimplyBlue SafeStain #LC6065, Invitrogen (Carlsbad,

CA, USA)

31

Sodiumhydroxid #71690, Fluka (St. Louis, MO,

USA)

Streptavidin:biotinylated alkaline phosphatase

Complex (5µg/ml) #F014-62, BioSpa (Italy)

Streptavidin-HRP #RPN1231V,

AmershamBiosciences


Streptavidin Sepharose (high performance) #17-5113-01,

AmershamBiosciences


Sulfo-NHS-LC-Biotin #21335, Pierce (Rockford, IL,

USA)

Sulfo-NHS-SS-Biotin #21331, Pierce (Rockford, IL,

USA)

Sypro Ruby #S-21900, Molecular Probes

(Invitrogen, Carlsbad, CA,

USA)

Thiourea #T-7875, Sigma (St. Louis, MO,

USA)

Trichloroacetic acid (TCA) #91230, Fluka (St. Louis, MO,

USA)

Trifluoracetic acid (TFA) #91699, Fluka (St. Louis, MO,

USA)

Tris #17-1321-01, plusone

AmershamBiosciences


Trypsin #V5111, Promega (Medison,

Seoul, Korea)

Trypsin porcine pancreas #T0303, Sigma (St. Louis, MO,

USA)

Trypsin/EDTA solution #25300-054, Gibco (Invitrogen


USA)

32

Tween 20 #P-1379, Sigma (St. Louis, MO,

USA)

Urea #17-1319-01, plusone

AmershamBiosciences


X-Ray film #165 1454, KODAK Xar-5

(Stuttgart, Germany)

Xylazin Vet. Streuli (Uznach, Switzerland)

2.1.2 Laboratory material

Anatomic tweezers fine 14.5cm #07.320.14, Polymed


Anatomic tweezers very fine 10.5cm #61.064.10, Polymed


Hg-Blood pressure measurement instrument,

Miniatur 300b in aluminium box #65-11-189, Speidel und

Keller (Zürich, Switzerland)

Cell culture flasks with filter caps

T-150 #90151, TPP (Trasadingen,

Switzerland)

Cannulae for FPLC sample application 2R2

0.7 x 50 mm #13.205, Unimed (Lausanne,

Switzerland)

Cell scrapers #9903, TPP (Trasadingen,

Switzerland)

Chirurgic scissors straight acute/acute 12cm #07.220.12, Polymed


Cotton swabs 15 cm, sterile #IVF1625300, Polymed


Cryotube #V7384, Sigma (by Nunc) (St.

Louis, MO, USA)

33

0.5 ml Eppendorf tube #72.735.002, Sarstedt

(Nürnbrecht, Germany)

1.5 ml Eppendorf tube (safe-lock) #0030 120.086, Eppendorf-

Netheler-Hinz-GmbH

(Hamburg, Germany)

2 ml Eppendorf tube (safe-lock) #0030 120.094, Eppendorf

Netheler-Hinz-GmbH

(Hamburg, Germany)

Falcon tubes

15 ml #91015, TPP (Trasadingen,

Switzerland)


Switzerland)

Feather Microtome Blade Carbon Steel C35 #FEATHER-C35, MICROM

(Walldorf, Germany)

Fibre scissors straight acute/acute 10cm #61.127.12, Polymed


FPLC adaptor fittings kit #732-0112, BioRad (Hercule,

CA, USA)

Gloves #E330, Kimberly-Clark

(Rosswell, USA)

#8F1320, SemperMed (Wien,

Austria)

Halssted-Mosquito Hemstatic forceps, straight,

12.5cm #13008-12, Fine Science Tools

(Heidelberg, Germany)

Heidelberger extension 30 cm #03.850.08, Polymed


Hydroxyapatite chromatography column:

Econo-Pac CHT-II Catridge #732-0083, BioRad (Hercule,

CA, USA)

Infusion canteen „Intrafix-Air“ 180cm #03.850.05, Polymed


34

Luer-Lock Dreiweghahn “Discofix” with extension #03.850.17, Polymed


Luer-Lock Dreiweghahn “Discofix” #03.850.15, Polymed


Medinop protection requirement 39 x 45 cm #IVF8760700, Polymed


Mesoft compresses 10 x 10 cm #SCA 156315, Polymed


Microcon Ultrafree-MC centrifugal filter devices (5µm) #UFC30SV00 Millipore


Microcon centrifugal filter devices

MWCO: 3 kDa

#42403, Millipore (Billerica, MA,

USA)

MWCO: 10 kDa

#42406, Millipore (Billerica, MA,

USA)

Micro-Mosquito Hemostatic forceps, curved,

12cm #13011-12, Fine Science Tools


Micro- tubing #232982, PTFE-tube, (ID:

0.5mm), Maagtechnic

(Dübendorf, Germany)

Molinea Plus-D 20 x 40 cm #IVF1608905, Polymed






One-way cannulae 26G/12mm #03.403.23, Polymed


35

0.2 ml PCR-Tubes #1044-00-0, Robbins (Asbach,

Germany)

0.2 ml PCR-Tube caps #1044-12-0, Robbins (Asbach,

Germany)

PD-10 Columns prepacked with

Sephadex G-25 medium #17-0851-01, Amersham

Bioscienses (Buckinghamshire,

GB)

Perfusion cannulae By Martin Mörser, Institute for

LTK

Pipette tips:

Gel loading tips #010-R204, QSP (Atlanta, GA,

USA)

200 µl #70.760.502, Sarstedt


1000 µl #70.762.100, Sarstedt


Biosphere Filter tips 1000µl #70.762.211, Sarstedt


Pyrex borosilicate glass #14832, Conrining (New York,

NY, USA)

Resin embedding mold 10 x 20 mm #23244, Polysciences

(Warrington, Pennsylvania,

USA)

Schwartz micro-serrefines vascular clamps,

straight, 12.5cm #18052-01, Fine Science Tools


Serological pipettes:


Switzerland)


Switzerland)


Switzerland)

36

25 ml #4489, Corning (Acton, MA,

USA)

50 ml #4490, Corning (Acton, MA,

USA)

StarFrost adhesive slides, Knittelgläser 24 x 50mm # BB024050A1

Menzel (Braunschweig,

Germany)

Steril filters:

250 ml #83.1822.001, Sarstedt


500 ml #83.1823.001, Sarstedt


Student Vannas Spring Scissors #15100-09, Fine Science Tools


Superfrost/Plus glass microslides #J1800AMNZ, Menzel

(Braunschweig, Germany)

Surgical Scissors “Lexer Baby”, straight, 10 cm #14078-10, Fine Science Tools


Syringe 10 ml Luer-Lok #300912, BD (Franklin Lakes,

NJ, USA)

Syringe 5 ml Luer-Lok #300911, BD (Franklin Lakes,

NJ, USA)

Vivaspin 20 (MWCO: 10kDa) (concentrator) #VS2002, VIVASCIENCE

(Hannover, Germany)

Vivaspin 500 (MWCO: 10kDa) (concentrator) #VS0101, VIVASCIENCE

(Hannover, Germany)

96-well plate #3363, Corning (Acton, MA,

USA)

96-well lid #3092, Corning (Acton, MA,

USA)

ZipTip C18 (P10) #ZTC18S960, Millipore


37

2.1.3 Instruments

Balances Weight resolution 0.1 mg AT460, Mettler Toledo

(Columbus, OH, USA)

Weight resolution 10 mg PE3600, Mettler Toledo

(Columbus, OH, USA)

Cambridge electrophoresis apparatus EV200, Protein Gel System

large Format PAGE Unit

Invitrogen (Carlsbad, CA, USA)

Camera connected to the microscope C5985, chilled CCD camera,

Hamamatsu (Hamamatsu city,

Japan)

Centrifuges:

For eppendorf tubes 5415D, Eppendorf centrifuge,

Netheler-Hinz-GmbH

(Hamburg, Germany)

For falcon tubes and 96-well plates Megafuge 1.0R, Heraeus

instruments, Kendro laboratory

products (Zürich, Switzerland)

Cryostat HM 505N, MICROM (Walldorf,

Germany)

fridge Liebherr turbo fresh,

Burkhalter+Co. (Wallisellen,

Switzerland)

-20°C freezer Liebherr Premium


Switzerland)

-86 °C freezer Model 923, Forma scientific,


Switzerland)

Film developer Curix 60, AGFA (Mortsel,

Belgium)

38

Fladbed scanner 1236u, SNAPSCAN, AGFA

(Mortsel, Belgium)

FPLC apparatus ÄKTA FPLC, Amersham

Bioscienses (Buckinghamshire,

GB)

Gel imaging Diana III, Raytest

Isotopenmessgeräte

(Straubenhardt, Germany)

Homogenisator Ultra-Turrax T8, Faust

Laborbedarf AG (Schaffhausen,

Switzerland)

37 °C incubator for 96-well plates wtc binder, rich-mond (Wigan,

GB)

Incubator Model 3111, Serie II, water

jacked CO2

incubator with HEPA filter,

ThermaForma (USA)

Laminar airflow cabinet 2000, Faster Cytosafe

Liquid nitrogen tank Cryo biological storage

system Locator 8plus,

Thermoline, Smithfield

(Australia)

Magnetic stirrer Ikamag RTC, Renner Gmbh

(Ludwigshafen, Germany)

Mass spectrometer Finnigan LCQ DECA

Microplate reader VERSAmax tunable, Molecular

Devices

Microscope (for histochemistry) Axiovert S100 TV and Axiomat

2 Mot Plus equipped with a

Polaroid DMC Digital

Microscope Camera, Carl-Zeiss

(Baden-Württemberg,

Germany)

39

Microscope (cell culture) Axiovert 25, Carl-Zeiss (Baden-

Württemberg, Germany)

MilliQ producing apparatus RiOs 5 and Milli-Q RG,

Millipore (Billerica, MA, USA)

Orbital shacker Unimax2010, Heidolph

Instruments (Cinnaminson, NJ,

USA)

Overhead rotator

pH-Meter PB-20, Sartorius (Göttingen,

Germany)

Pipette boy IBS Pipetboy acu, INTEGRA

Biosciences (Chur,

Switzerland)

Pipettes:

2 – 20 µl Calibra 822, Socorex

(Lausanne, Switzerland)





Multichannel 20 – 200 µl Calibra 852, Socorex


Power supply to Cambridge electrophoresis app. E143 Consort,

AmershamBiosciences


Sonication bath Sonorex RK100, Bandelin

(Berlin, Germany)

Speed vac SC110A, Savant SpeedVac

plus, GMI, (Alberville,

Minestota, USA)

Syringe 701N 80383/00, Microliter

Syringes, Hamilton (Reno, NV,

USA)

Waterbath

40

41

(37 °C) Salvis

(56 °C) TE-10A, Techne, Bender und

Hohlbein AG (Zürich,

Switzerland)

Vortex Si Vortex Genie 2\

XCell II Blot Module #EI9051, Invitrogen (Carlsbad,

CA, USA)

XCell SureLock Mini-Cell #EI0001, Invitrogen (Carlsbad,

CA, USA)

2.1.4 Solutions and Buffers

PBS (Phosphate buffered saline)

20 mM NaH2PO4

30 mM Na2HPO4

100 mM NaCl

in MilliQ

Further solutions and buffers used only for one method are specified in the

corresponding method chapter.

2.2 Methods 2.2.1 Implantation of F9 teratocarcinoma allografts in SvEv129 mice Animal experiments were approved by the Swiss Federal Veterinary Office (license

83/2002 Supplement) and performed in accordance with the Swiss Animal Protection

Ordinance.

2.2.1.1 Solutions and buffers DMEM / FCS / AB (Cell culture medium per F9 cells) 100 % (v/v) Dulbecco’s Modified Eagle Medium (DMEM) (+4500mg/L

Glucose+LGlutamin and -Pyruvate)

10 % (v/v) Foetal bovine serum (10%)

1 % (v/v) Antibiotic-Antimycotic (containing Penicillin G sodium, Streptomycin

sulfate and Amphotericin B) (1%)

Medium for the cryopresevation of F9 cells 20 % (v/v) Foetal bovine serum (FCS)

10 % (v/v) Dimethylsulfoxide (DMSO)

70 % (v/v) Dulbecco’s Modified Eagle Medium (DMSO) with 1% Antibiotic-

Antimycotic

0.1% gelatine solution 5 % (v/v) Gelatine 2% gelatin solution (Typ B: From Bovine Skin)

95 % (v/v) MilliQ

(Sterilisation by filtration)

42

2.2.1.2 F9 cell culture F9 cells were cultured in cell culture flasks with a surface area of 150 cm2. The flasks

were coated previously with 5 ml of a 0.1% gelatine solution at 37°C for at least 20

min and washed with 10 ml PBS. 25 – 35 ml F9 cell culture medium were used per

flask. For seeding, cryopreserved cells were thawn in a 37°C water bath for 1-2

minutes, resupended and dispersed in the medium. Cells were grown in a humidified

atmosphere containing 5% CO2 at 37°C. 24 hours after seeding the medium was

renewed to eliminate the cryomedium.

When reaching 70 - 90% confluency, cells were splitted as follows: The medium was

removed. Cells were washed with 10 ml PBS and detached by incubation with 3 ml

Trypsin / EDTA solution for 1-2 minutes at 37°C. 3 ml medium were added to stop

proteolysis. This cell suspension was distributed among new flasks. In general, cells

from a confluent flask splitted 1:2 reached confluency again after ~24 h.

Part of the cells was expanded for cryopreservation. From one cell culture flask

grown to 70-90% confluency three cryo tubes were prepared as follows: The medium

was removed. Cells were washed with 10 ml PBS and detached with Trypsin/EDTA

as described before. After addition of 3ml F9 cell culture medium, the cells were

pelleted (Megafuge 1.0R, 1100 rpm, 4°C, 5 minutes), resuspended in 3ml cryo

medium and distributed to three aliqotes in cryo tubes. The cryotubes were frozen

immediately at -80°C for at least three day, then transferred to a liquid nitrogen tank.

43

Fig. 2. 1 F9 cells in culture

2.2.1.2 Injection of mice with F9 cells When cells reached 70 – 90% confluency the medium was removed. For the injection

of five mice one cell culture flask was processed as follows: Cells were washed with

10 ml PBS and detached with Trypsin/EDTA as described before. After addition of

3ml F9 cell culture medium, the cells were pelleted (Megafuge 1.0R, 1100 rpm, 4°C,

5 minutes). The supernatant was discarded and the cell pellet was resupended in 0.5

ml PBS. The cell suspension was stored on ice and immediately used for injection.

Female SV129 mice received subcutaneous injections of 100 µl cell suspension (~3-

4 x 106 cells). The mice were monitored regularly, and the tumor volume was

measured with a caliper, using the formula “volume = length x width2 x p/6”. When

showing any sign of pain or suffering, when the tumor size exceeded 10% of the

body weight, or in case of a body weight loss >15% animals were euthanized.

Tumor-bearing mice were used for perfusion experiments when the tumor had

reached a size of ~200 – 1200 mm3.

Fig. 2.2 SvEv129 mouse bearing an F9 teratocarcinoma allograft

44

2.2.2 In vivo biotinylation 2.2.2.1 Solutions and buffers Perfusion solution 10% (w/v) Dextran

In PBS

(Filtration before using)

Biotinylation solution 1 mg/ml Sulfo-NHS-LC-Biotin

alternatively

1.09 mg/ml Sulfo-NHS-SS-Biotin

In perfusion solution

(Preparation just before the surgery)

Quenching solution 50 mM Tris

10 % (w/v) Dextran

In PBS


Sprinkling solution 50 mM Tris

In PBS


Anaesthesia mixture 200 mg / kg body weight Ketamin (for anaesthesia)

20 mg / kg body weight Xylazin (as analgesic and relaxant)

3 mg / kg body weight Azepromazin (as tranquilizer)

In 0.9 % NaCl

45

2.2.2.2 Anaesthesia For perfusion experiments, mice were anaesthetized by a combined subcutaneous

injection of Ketamine, Xylazin and Acepromazine. The dose was adapted

appropriately (about 200-300 mg/kg Ketamin, 20 mg/kg Xylazin and 3 mg/kg

Acepromazine) and administered by injection of 10µl of a premixed anaesthesia

cocktail per g body weight. It took 5 to 20 minutes until the narcosis acted. The

reflexes were checked to make sure that the animal was well anaesthetized. When

the reflexes were still present after 20 minutes the mouse was injected again with

approximately half of the original dose.

2.2.2.2 Surgery The anaesthetized mouse was fixed with adhesive tape onto an inclined plastic shelf

which was covered with an absorbent incontinence pad. The fur was wetted with

alcohol to avoid hairs in the surgery field. The skin was cut under the sternum with

blunt scissors, a cut was made up to chest and the skin was dissected. The thorax

was opened through a median sternotomy. The heart was hold with tweezers,

carefully lifted and slightly turned enabling to puncture the left heart ventricle with a

perfusion needle. The perfusion needle was a cannula of different size (depending on

the experiment, see table in 2.2.2.3), which was provided with a barb by Martin

Mörter (Institute of Laboratory Animal Sciences, University of Zurich). A small cut was

made in the right atrium to allow the outflow of the perfusate. At this time, the mouse

was readily prepared ( see Fig. 2.3) and the perfusion was started immediately.

46

Fig.2.3 Picture of a mouse which is

prepared for perfusion (Note:The photo was taken after perfusion and preparation of anatomic structures and kindly provided by Dr. Christian Matter, Institute of Physiology and University Hospital, University of Zurich.)

2.2.2.3 Perfusion After surgery, the large circulation of the mouse was perfused from the left heart

ventricle (LV) to the right atrium (RA) according to the scheme in Fig. 2.4. and

applying the conditions specified in Tabl. 2.1.

47

100 1. mmHg 3.Washing solution

(optional)

Lung capillaries Quenching solution

RA

LA

LV RV

2.Biotinylation

Systemic solution capillaries

Tumor

Fig. 2.4 Scheme of the perfusion procedure LA: Left atrium

LV: Left ventricle RA: Right atrium RV: Right ventricle (See text for further explanations.)

The pressure was monitored by means of a manometer connected via a washing

flask to the perfusion tube system, and was kept constant at 100 mm Hg. The

biotinylation reagent was stored in aliquots in argon at -20°C and dissolved

immediately before the biotinylation step. Depending on the experiment, the

perfusion was performed in 2 or 3 steps with different perfusion solutions:

1. Step (optional): Blood and cells were washed away with a washing solution.

2. Step: The actual in vivo biotinylation step was the perfusion with the

biotinylation solution containing a charged, activated ester

derivative of biotin, which cannot diffuse through biological

membranes. Proteins, glycolipids and phospholipids carrying

48

accessible primary amino groups might be labeled in this

process.

3. Step: To neutralize unreacted biotinylation reagent, the in vivo

biotinylation was followed by a washing step with a quenching

solution containing primary amino groups.

The composition of the perfusion solutions and further perfusion conditions were

different in the individual experiments presented in this thesis and are specified in

table 2.1.

The perfusion flow rate depended on the size of the perfusion cannula used and on

the viscosity and therefore of the composition of the perfusion solution. In the

experiments with warmed perfusion solutions, the solutions were incubated in a 38°C

water bath before perfusion. As for the biotinylation solution, the biotinylation reagent

stored at -20°C was dissolved in a 38°C pre-warmed solution immediately before

use. In the indicated cases, the anaesthetized animal was warmed during surgery

and perfusion using a heating pad, which was placed between the plastic shelf and

the absorbent incontinence pad.

2.2.2.4 Preparation of specimens

After perfusion, organs and tumors were immediately excised and specimens were

either freshly snap-frozen for the preparation of organ homogenates or embedded in

cryo-embedding compound and frozen in liquid nitrogen-chilled isopentane for the

preparation of cryo-sections for histochemical analysis.

49

Pe

rfusio

n ne

edle

Pe

rfusio

n pr

essu

re

Perfu

sion

flow

rate

Tem

pera

ture

of

perfu

sion

solu

tions

War

min

g th

e an

imal

1. P

erfu

sion

step

: wa

shin

g

2. P

erfu

sion

step

: bio

tinyla

tion

3. P

erfu

sion

step

: qu

ench

ing

Orig

inal

pro

toco

l 25

G b

utte

rfly

with

sm

all d

iam

eter

tu

be

100

mm

Hg

~1

.5 m

l/min

RT

No

10

min

PB

S

10 m

l Su

lfo-N

HS-L

C-bi

otin

1

mg/

ml

in P

BS

10 m

in

50 m

M T

ris

in P

BS

High

flow

(3

.1.1

) m

iscel

lane

ous

(see

tabl

e 3.

1)

100

mm

Hg

m

iscel

lane

ous

(see

tabl

e 3.

1)

RT

No

10 m

in

PBS

misc

ella

neou

s (s

ee ta

ble

3.1)

10 m

in

50 m

M T

ris

in P

BS

Dext

ran

as

plas

ma

expa

nder

(3

.1.3

)

25G

with

out s

mal

l tu

be

100

mm

Hg

~1

.8 m

l / m

in

RT

No

No

15 m

l Sul

fo-

NHS-

LC-B

iotin

1

mg/

ml,

10%

(w/v

) De

xtra

n 40

in

PBS

pH

7.4

10 m

in

50 m

M T

ris,

10%

(w/v

) De

xtra

n 40

in

PBS

In v

ivo

biot

inyla

tion

with

Su

lfo-N

HS-S

S-Bi

otin

(3

.4)

25G

with

out s

mal

l tu

be

100

mm

Hg

~1

.8 m

l / m

in

38°C

He

atin

g pa

d an

d IR

-la

mp

No

15 m

l Sul

fo-

NHS-

SS-B

iotin

1.

09 m

g/m

l, 10

% (w

/v)

Dext

ran

40

in P

BS

pH 7

.4

10 m

in

50 m

M T

ris,

10%

(w/v

) De

xtra

n 40

in

PBS

Fina

l opt

imize

d pr

otoc

ol

(3.1

.3)

25G

with

out s

mal

l tu

be

100

mm

Hg

~1

.8 m

l / m

in

38°C

He

atin

g pa

d an

d IR

-la

mp

No

15 m

l Sul

fo-

NHS-

LC-B

iotin

1

mg/

ml,

10%

(w/v

) De

xtra

n 40

in

PBS

pH

7.4

in P

BS

10 m

in

50 m

M T

ris,

10%

(w/v

) De

xtra

n 40

in

PBS

Tabl. 2.1 Different perfusion conditions of the individual experiments. Numbers in brackets indicate the corresponding chapter.

50

51

2.2.3 Perfusion and injection of mice with Hoechst dye

To compare the accessibility of vascular structures for a molecule either by

intravenous injection or by perfusion we tested both applications with a nuclei-

staining fluorescent dye and compared staining patterns of organ sections. Mice

received an intravenous injection of 100 µl 1 mg/ml Bisbenzimide H 33342 (Sigma) in

an aqueous solution of 0.9% (w/v) NaCl. In a simplified calculation and with an

assuming a blood volume of 2 ml, this injection corresponded to a total concentration

of 0.05 mg/ml in the blood. After 2 or 4 min the animal was sacrificed. Other mice

were anaesthetized, underwent surgery as described in 2.2.2.2 for the in vivo

biotinylation and were perfused with 0.05 mg/ml Bisbenzimide H 33342 in an

aqueous solution of 0.9% (w/v) NaCl for 4 min. A washing perfusion step with 0.9%

NaCl solution was either performed for 10 min prior or for 6 min after the perfusion

with the fluorescent dye to test the impact of the previous wash step. Immediately

after sacrifizing the injected animals or after perfusion respectively, organs were

excised, embedded in cryoembedding compound and frozen in isopentane in liquid

nitrogen. 10 µm sections were cut using a Microm HM 505N cryostat, placed onto

Superfrost Plus glass microslides and analyzed with an Axiomot 2 Mot Plus

microscope using filter set 02 and the Axiovision software.

2.2.4 Histochemistry 2.2.4.1 Buffers and Solutions TBS 100 mM NaCl (mM)

50 µM Tris

In MilliQ

Buffer A 0.01 %(w/v) Aprotinin

In TBS

Buffer C 2 mM MgCl2

In TBS

Levamisole solution 24.1 % (w/v) Levamisole

In 100mM Tris-HCl (pH 8.2)

Fast-Red TR solution 98 % (v/v) 100mM Tris-HCl (pH 8.2)

2 % (v/v) N,N-Dimethylformamid

0.02 % (w/v) Naftolol ASMX Phosphat

0.0241 % (w/v) Levamisole solution

0.04 % (w/v) MgCl2

2.2.4.2 Cutting of cryosections 10 µm sections of frozen (at -80° C) tissues embedded in cryomedium were cut with

a cryostat at -15° C and placed on Superfrost/Plus glass microslides. The sections

were dryed for 30 to 120 minutes at 37° C, fixed by immersion in chilled acetone for

10 minutes and dryed on air. The sections were immediately used for staining or

stored at -80° C.

52

53

2.2.4.3 Histochemical staining with Streptavidin:biotinylated alkaline phosphatase

The acetone fixed sections were rehydrated in buffer A for 5 minutes. The sections

were incubated for 30 minutes with FCS, washed twice with buffer C for 5 minutes,

and incubated with streptavidin:biotinylated alkaline phosphatase complex (diluted

1:200 in buffer C) for 30 minutes. After washing 4 times with buffer C the Fast-Red

TR powder was dissolved in the Fast-Red TR solution at a concentration of 1 mg/ml.

The slides were incubated with the prepared, filtered Fast-Red TR solution for about

15 - 20 min to develop coloration. The reaction was stopped by immersing the

sections in water and the sections were counterstained by immersing in filtered Harris

Haematoxilin for 2 - 5 minutes. After washing twice with water the sections were

mounted with mounting medium and covered with a cover slip. The slides were

analysed microscopically. Pictures of the slides were taken with a Polaroid DMC

Digital Microscope Camera using the Axiovision software. All image files were

exported to Adobe Photoshop.

2.2.5 Preparation of protein extracts from tissue specimens 2.2.5.1 Buffers and solutions Lysis buffer 10 mM EDTA

1 tablet / 50ml Complete EDTA free proteinase inhibitor cocktail

2 % (w/v) SDS

50 mM Tris

In PBS (pH6.8)

2.2.5.2 Tissue homogenisation Organ specimens stored at -80°C were transferred each to a 5 ml test tube

andmogenised with 20 µl lysis buffer per mg tissue using an Ultra Turax T8 disperser

applying six intervals of 20 seconds full power and 20 seconds standby at moderate

cooling on ice. The homogenates were stored in aliquots in 1.5ml safe lock

Eppendorf tubes at -80°C.

2.2.5.3 Protein extraction The frozen organ homogenates were thawed at room temperature and processed by

5 minutes sonication (to shear DNA), 5 minutes incubation at 99°C and 5 minutes

centrifugation at 16’100 x g (see Fig. 2.5). The resulting supernatant was taken as

total protein extract. Protein extracts were stored in aliquots at -80°C.

54

organ homogenates

55

thawn at RT

5 min sonication bath (to shear DNA)

spinned down

99°C for 5 min

cooled to

Centrifuged 5 min at 16100 g

Protein determin.supernatant used as total protein extract

Fig. 2.5 Overview of the protein extraction procedure

2.2.5.4 Protein determination Protein concentration of the extracts was determined using the BCA Protein Assay

Reagent Kit from Pierce according to the instruction manual (microplate procedure

version).

Data were processed by SIGMAPLOT 8.0 and the MICROSOFT EXCEL software.

2.2.6 Purification of biotinylated proteins To reduce the complexity of a protein extract from in vivo biotinylated mice the

biotinylated proteins were purified on streptavidin sepharose according to the

following scheme:

SA- Sepharose

Releasing

SUPSUP

B

INPUT

+

REL Centrifugation

B

Capturing

CONCENTRATION

SPIN DIALYSIS

B

Centrifugation

Washing

SA- Sepharose

Releasing

SUPSUP

B

INPUT

+

REL Centrifugation

B

Capturing

CONCENTRATION

SPIN DIALYSIS

B

Centrifugation

Washing

Protein

Biotin

Streptavidin sepharose

Protein

Biotin

Streptavidin sepharose

Incubation and working steps were performed in 1.5 ml Eppendorf tubes. (Exception:

In the experiments described in 3.3 (on resin digestion) the steps were performed in

centrifugal filter devices.) After all washing and incubation steps, the resin was

pelleted by centrifugation (30 seconds at 800 x g).

Depending on the experiment one of the following procedures was chosen:

Parameters 1)

Original protocol (3.2.1)

2) Concentration on

resin (3.2.3)

3) Sample

concentration by spin dialysis

(3.2.4)

4) Tryptic on resin

digestion (3.3)

SA- sepharose slurry (µl)

320 160 640 100

Washing buffers (A, B, C) (µl)

500 500 1000 500

Elution buffer (µl) 500 150 1000 _ Annotation Washing steps

performed in centrifugal filter

devices (diameter: 5 µm)

Fig.2.6 Overview of the purification of biotinylated proteins by SA sepharose

56

2.2.6.1 Buffers and solutions Wash solutions Buffer A (1) 1 % (w/v) NP-40

0.1% (w/v) SDS

In PBS

Buffer A (2) 1% (w/v) NP-40

2% (w/v) SDS

In PBS

Buffer B 0.4 M NaCl

In buffer A

Buffer C (1) 50 mM Tris

In MilliQ

Buffer C (2) 50 mM Tris in MilliQ

2 % (w/v) SDS

In MilliQ

Elution NaOH (8M) 8M Natriumhydroxid

In MilliQ

Biotin stock solution 302mM D-Biotin

57

58

8 % (v/v) NaOH (8M)

In MilliQ

(pH ~13.1)

Elution solution 2 M Thiourea

6 M Urea

1% v/v) Biotin stock solution

2 % (w/v) SDS

ad 10ml PBS

2.2.6.2 Capture SA sepharose was washed twice with buffer A and twice with PBS. The protein

extract (referred as INPUT) was mixed with the SA sepharose. Biotinylated proteins

were captured for 2 h at room temperature on an overhead rotator.

2.2.6.3 Washing To eliminate unspecifically bound proteins, the reaction mixture was pelleted by

centrifugation, washed 3 times with bufferA, twice with buffer B and once with buffer

C.

2.2.6.4 Elution The biotinylated proteins were eluted by incubation with elution solution and

incubated for 15 min at room temperature and 15 min at 96 °C.

2.2.7 Sample concentration 2.2.7.1 Spin dialysis 2.2.7.1.1 Microcon centrifugal filter devices

Fig. 2.7 Microcon centrifugal filter devices

Up to 500 µl of the sample were filled into the sample reservoir of the Microcon

centrifugal filter devices (3 kDa or 10 kDa). The filter devices were centrifuged at

14’000 x g until the desired volume reduction was reached. The sample reservoir was

placed upside down in a new vial and centrifuged for 3 minutes at 1000 x g to collect

the concentrate.

2.2.7.1.2 Vivaspin 500 concentrator

Fig. 2.8 Vivaspin 500

concentrator

59

60

Up to 500 µl sample were filled into the sample reservoir of the Vivaspin 500

concentrators (10 kDa). The concentrators were centrifuged at 12’000 x g (up to

15’000 x g) until the desired volume reduction was reached. The concentrate was

collected with a 200µl pipette and was transferred to a 1.5 ml safe-lock Eppendorf

tube.

2.2.7.2 TCA precipitation 100 µl of 100 % (w/v) TCA were added to 1 ml sample containing at least 5 µg

protein. The sample was vortexed and allowed to precipitate for 15 minutes at -20°C.

After centrifugation (for 5 minutes, at 10’000 x g) the supernatant was discarded and

the pellet was washed with 1 ml Ethanol / Ether (1:1) before resuspending in 24 µl

hydroxyapatite chromatography elution buffer A or elution buffer B.

2.2.8 Protein fractionation by hydroxyapatite chromatography Hydroxyapatite chromatography was used for the fractionation of the protein extract

derived from in vivo biotinylated mice. The hydroxyapatite chromatography was performed with a pre-packed catridge from

Bio-Rad. The FPLC-system was equipped with an 1-ml-injection loop and controlled

with the software UNICORN (Version 3.1). An additional 2 ml tubing was installed in

front of the detector to be able to also collect the dead volume.

2.2.8.1 Buffers and solutions Sample preparation solution 50 mM Tris

100mM DTE

1% (w/v) SDS

1 tablet / 50ml Complete EDTA free proteinase inhibitor cocktail (50x)

In MilliQ

(alternatively: without Complete EDTA free proteinase inhibitor cocktail (50x))

Standard protein mixture solution (1) 0.96 mg/ml Pepsin

0.26 mg/ml Fibrinogen

0.26 mg/ml Trypsin

0.02 mg/ml Lysozym

In sample preparation solution

Standard protein mixture solution (2) 0.96 mg/ml Pepsin

0.48mg/ml Fibrinogen

0.48mg/ml Trypsin

0.48mg/ml Lysozym

61

In sample preparation solution

Phosphate stock solutions Base A 0.1 M HNa2PO4

In MilliQ

Acid A 0.1 M H2NaPO4

In MilliQ

Base B 1M HNa2PO4

In MilliQ

Acid B 2.5 M H2NaPO4

In MilliQ

Washing solution A 0.1 mM CaCl2

7 % (v/v) base A

3% (v/v) acid A

In MilliQ

(pH adjustment with HCl and NaOH, pH 7.2)

(alternatively without CaCl2)

Washing solution B 1 % (w/v) SDS

7.5 µM CaCl2

62

35 % (v/v) base B

6 % (v/v) acid A

In MilliQ



Elution buffer A (1) 1 % (w/v) SDS

0.1mM CaCl2

0.5mM DTE

3% acid A

7% base A

(pH adjustment with HCl and NaOH)

Elution buffer A (2) 1 % (w/v) SDS

50 mM Tris

0.5 mM DTE

in MilliQ

(pH adjustment with HCl and NaOH, pH7.2)

Elution buffer B (1) 1 % (w/v) SDS

7.5 nM CaCl2

0.5 mM DTE

35 % (v/v) base B

6 % (v/v) acid A

In MilliQ

(pH adjustment with HCl and NaOH)

63


Elution buffer B (2) 1 % (w/v) SDS

0.5 mM DTE

42 % (v/v) base B

7.2% (v/v) acid A

In MilliQ


Washing solution C 1.5% (v/v) acid A

3.5% (v/v) base B

In MilliQ


Storage solution 20% (v/v) Ethanol

80% (v/v) Washing solution (used before catridge storage, pH 7.0)

2.2.8.2 Washing of the column The flow rate was set to 0.8 ml/min. Before each run the catridge was washed for 3

minutes with filtered washing buffer A and for 10 minutes with filtered washing buffer

B. The inverted catridge was equilibrated with buffer A for 15 minutes.

Fig. 2.9 Pre-packed CHT II catridge (BioRad) used fort he hydroxyapatite chromatography

64

2.2.8.3 Sample preparation For sample loading, 1.4 ml filtered (diameter: 0,22 µm) sample was injected into the

1ml-injection-loop.

Depending on the experiment a 10 min heating step at 96 °C was performed or not

and different protein amounts were loaded on the column:

Standard protein

mixture (unoptimized)

Standard protein mixture

(optimized)

Biotinylated kidney sample

Biotinylated RENCA sample

Loaded protein amount (mg )

3

1.6

0.5

1.6

2.2.8.4 Chromatography process The applied sample was eluted with an increasing phosphate concentration applying

a linear gradient of buffer B in buffer A (see Fig. 2.10). Before starting the gradient, it

was washed with a certain volume of buffer A (gradient delay).

0

1234

56789

1 2 3 4 5 6 7 8 9

Buf

fer

B [%

]

Elution volume [ml]

Linear gradient

0

1234

56789

1 2 3 4 5 6 7 8 9

Buf

fer

B [%

]

Elution volume [ml]

Linear gradient

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

Buf

fer

B [%

]

Elution volume [ml]

Part

1

Part 2

Segmented gradient

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

Buf

fer

B [%

]

Elution volume [ml]

Part

1

Part 2

Segmented gradient

Fig.2.10 Graphs showing the linear gradient (in red) and the segmented gradient (in green)

According to the experiment one of the following elution programs (1, 2, 3, 4 or 5)

were used:

65

66

Program 1 2 3 4 5 Column volume (ml) 1 1 1 1 1 Pressure limit (mPa) 0.3 0.3 0.3 0.3 0.3 Flow rate (ml/min) 0.8 0.8 0.8 0.8 0.8 Fraction size (ml) 1.6 2 2 1.5 1.5 Linear gradient Length (ml) 24 24 - 24 -

Percentage of buffer B at the end of the gradient (%) 100 100 - 100 -

Segmented gradient Length of gradient part 1 (ml) 6 3

Percentage of buffer B at the end of the gradient part 1 (%) 50 25

Length of gradient part 2 (ml) 18 21

Percentage of buffer B at the end of gradient part 2 (%) 100 100

Gradient delay (ml) 5 4 4 3 3

2.2.8.5 Catridge storage After use, the catridge was washed with washing solution C and stored in storage

solution. After deinstallation of the catridge, the FPLC system was stored in filtered

20 % Ethanol solution.

2.2.9 SDS-PAGE and Strepavidin blot 2.2.9.1 Buffers and solutions Loading buffer (5X) 210 mM Tris-HCl, pH 6.8

33.6 % (v/v) Glycerol

235 mM SDS

0.67 ‰ (w/v) Bromophenolblue

in MilliQ

Reducing loading buffer

0.05 % (v/v) β-Mercaptoethanol

In Loading buffer (5x)

MOPS running buffer 5 % v/v) 20 x MOPS SDS running buffer (1 M MOPS, 1 M Tris, 69.3 mM SDS,

20.5 mM EDTA)

In MilliQ

Transfer buffer 20 % (v/v) Methanol

5 % (v/v) 20 x MOPS SDS running buffer

In MilliQ

4 % M-PBS 4 % (w/v) Milk powder

In PBS

4 % M-PBS / Streptavidin-HRP solution 0.1 % (v/v) Streptavidin-HRP

In 4% M-PBS

0.1 % T-PBS 0.1 % (w/v) Tween 20

67

In PBS

Amersham ECL chemioluminiscens reagent 40 parts Reagent A

1 part Reagent B

2.2.9.2 SDS-PAGE 1D SDS-PAGE was performed using Invitrogen’s NuPAGE electrophoresis system

according to the instruction manual. Samples were mixed 4:1with 5 x concentrated

reducing or non-reducing SDS-loading buffer, heated for 10 minutes at 96° C and

loaded precast on NuPAGE NOvex Bis-Tris gels. Depending on the experiment one

of the following gels was chosen:

• 4 – 12 % Bis -Tris gel, 1.5 mm, 10 well

• 4 – 12 % Bis -Tris gel, 1.0 mm, 15 well

• 12 % Bis -Tris gel, 1.0 m, 15 well

Electrophoresis was carried out in an XCell SureLOCK Mini-Cell connected to a

powers cupply from Amersham using the following parameters:

Running buffer Voltage Maximal current Run time NuPAGE MOPS SDS Running buffer 180 V 110 mA for one gel 50 min

Fig.2.11 XCell SureLock Mini-Cell

68

2.2.9.3 Streptavidin blot For streptavidin blot analysis proteins were blotted from SDS-PAGE gels to

nitrocellulose membranes. SDS-PAGE was carried out as described above (2.2.9.2).

Protein blotting was performed using the XCell II blot Module and the XCell SureLock

Mini-Cell using the manual instruction. The following conditions were applied:

Transfer buffer Voltage Maximal current Run time 20 % Methanol in NuPage MOPS

SDS Running buffer 30 V 220 mA 90 min

After blotting the transfer membrane was blocked with 4 % (w/v) mik powder in PBS

(M-PBS) for at least one hour, rinsed twice with PBS and then washed twice for 5

minutes with PBS. The membrane was incubated with Streptavidin-Horseradish-

Peroxidase conjugate diluted 1:1’000 in M-PBS for 1 hour. After several washing

steps (2 x rinsing with PBS-Tween, 2 x washing with PBS-Tween for 5 minutes, 2 x

rinsing with PBS and 2 x washing with PBS for 5 minutes) proteins were detected

using the Amersham ECL+Plus Chemoluminescence kit (according to the instruction

manual): The membrane was incubated for 5 minutes in the dark with 2 ml of a

mixture of solution A and B in a ratio of 40:1 and KODAK BioMAX light film for 5

minutes. The film was developed with an AGFA Curix 60 automatic film developer.

Fig. 2.12 XCell II

blot module

69

2.2.10 Protein visualisation 2.2.10.1 Buffers and solutions Fixative solution 7 % (v/v) Acetic acid

10 % (v/v) Methanol

83 % (v/v) MilliQ

2.2.10.2 Gel staining 2.2.10.2.1 SyproRuby stain All steps were performed in plastic boxes covered with aluminium foil (photoresist) on

an orbital shaker at approximately 60 rpm. After electrophoresis gels were washed

with MilliQ and fixed in fixative solution for 30 min prior to incubation in Sypro Ruby

Gel Stain solution overnight (or for at least 3 hours). To decrease background

staining gels were subsequently washed with fixative solution for 10 minutes and with

MilliQ for several hours.

2.2.10.2.2 Simply blue Safe stain All steps were performed in plastic boxes covered with aluminium foil on an orbital

shaker at approximately 60 rpm. Gels were washed 3 times for 5 minutes with MilliQ

and stained in a mixture of 60 ml SimplyBlue SaveStain dye and 6 ml 20 % (w/v)

NaCl solution overnight. The gels were destained for at least one hour or overnight in

MilliQ.

70

71

2.2.10.3 Gel imaging 2.2.10.3.1 Sypro Ruby stained gels SyproRuby stained gels were imaged with a DIANA II chemiluminescence detection

system from raytest consisting of a cooled CCD-camera with 512 X 512 active pixel

connected to a computer. The camera was mounted on an evo III darkroom with

height adjustable UV light table. The gels were excited with UV light and

luminescence emission was recorded at 605 nm. Zoom and sharpness were adjusted

and with the best setting a picture was recorded.

2.2.10.3.2 SimplyBlue stained gels SimplyBlue stained gels were imaged using an AFGA SNAPSCAN 1236u flatbed

scanner with resolution of 600 x 1200 ppi. ScanWise was used as software. All image

files were exported to Photoshop.

2.2.11 Identification of proteins Proteins were identified by mass spectrometric analysis of the peptides resulting from

tryptic digestion.

Two different approaches were applied:

1) For the identification of proteins after separation by SDS-PAGE, protein bands

were excised from the gel and submitted to a tryptic in gel-digestion followed

by the extraction and analysis of the resulting peptides.

2) A “shot gun” approach was performed for the simultaneous identification of a

subset of proteins, separated by affinity capture on streptavidin sepharose

from a total protein extract of in vivo biotinylated mice tissues and submitted to

a tryptic “on-resin” digestion. The resulting peptides of this protein mixture

were analysed by LC-MS/MS without any further separation steps.

2.2.11.1 Buffers and solutions Trypsin stock solution 0.04 µg / µl Trypsin

In 50mM NH4HCO3 solution

Wetting solution 50 % (v/v) Acetonitrile

In MilliQ

Equilibration solution 0.1 % (v/v) TFA

In MilliQ

Sample preparation solution

72

0.1 % (v/v) TFA

In MilliQ

Elution solution 0.1 % (v/v) TFA

50 % (v/v) Acetonitrile

In MilliQ

Buffer A 0.5 % Formic acid

5 % Acetonitrile

94.5 % MilliQ

Buffer D 0.5 % Formic acid

99.5 % Acetonitrile

2.2.11.2 Sample preparation for µLC-MS/MS 2.2.11.2.1 Sample preparation starting from SDS-PAGE gel bands Sample preparation for mass spectrometric measurements from SDS-PAGE bands

consisted of cutting and washing of the gel pieces, proteolytic in-gel digestion of

proteins and extraction of the resulting peptides. All steps were perfomed according

to the protocol of the EMBL protein Peptide Group of 1997 with some modifications.

All steps were performed in 96-well plates using a multi-channel pipette and in a

sterile environment (laminar air flow) to prevent contamination.

All centrifugation steps were performed with the Megafuge 1.0R with 2’800 rpm at

4°C for 5 min., if not indicated otherwise.

73

• Excision of gel bands

• Reduction and alkylation(2.2.11.2.1.2)•Washing(2.2.11.2.1.3)•In-geldigestion withTrypsin(2.2.11.2.1.4)•Peptide extraction(2.2.11.2.1.5)

•µLC-MS/MS (2.2.11.3)

•Data evaluation 2.2.11.4

• Protein separation(SDS-PAGE)

(2.2.11.2.1.1)

• Excision of gel bands• Reduction and alkylation(2.2.11.2.1.2)•Washing(2.2.11.2.1.3)•In-geldigestion withTrypsin(2.2.11.2.1.4)•Peptide extraction(2.2.11.2.1.5)

•µLC-MS/MS (2.2.11.3)

•Data evaluation 2.2.11.4

• Protein separation(SDS-PAGE)

(2.2.11.2.1.1)

Protein IdentificationProtein Identification

Fig. 2.13 Schematic overview of the in-gel digest and the following

analysis with µLC/MS-MS

2.2.11.2.1.1 Excision of gel bands Protein bands from Sypro Ruby stained gels were cut in the open evo II darkroom on

the UV light table from the DIANA II system. The gel was constantly humidified with

MilliQ to prevent desiccation. Bands were excised by means of a sterile pipette tip.

Cut pieces were transferred to a 96-well microtiter plate, in which all their further

treatments were performed.

2.2.11.2.1.2 Reduction and alkylation The gel pieces were washed with 150 µl MilliQ for 5 min, centrifuged and the liquid

was removed. 180 µl acetonitril were added followed by pipetting three times up and

down in order to shrink the gel pieces until they turned white,. The samples were

centrifuged, liquid was removed and the shrunk gel pieces were dried in the vacuum

concentrator. Dried gel spots were rehydrated and incubated for 30 min at 56° C in

74

50 µl of a solution of 10 mM DTE and 100 mM NH4HCO3 in MilliQ in order to reduce

the proteins. After centrifugation, liquid was removed and the pieces were again

shrunk in acetonitrile. The acetonitrile was replaced by 50 µl of a solution containing

55 mM iodoacetamide in 100 mM NH4HCO3. After alkylating the proteins for 20 min

at room temperature in the dark, the iodoacetamide solution was removed and the

gel particles were washed with 150 to 200 µl 100 mM NH4HCO3 for 15 min. Samples

were spun down followed by replacement of the supernatant by 180 µl acetonitrile to

shrink spots. After centrifugation the acetonitrile was removed and the gel particles

were dried in the vacuum concentrator prior to the following washing procedure.

2.2.11.2.1.3 Washing After reduction and alkylation, samples were washed with 80 µl 100 mM NH4HCO3

for 5 min. An equal volume of 100 % acetonitrile was added and the samples were

incubated for 5 min on an orbital shaker. The gel pieces were spun down and the

supernatant was removed. This washing step was repeated twice.

Subsequently, the gel pieces were shrunk in 150 µl acetonitrile for 5 min on the

orbital shaker (~ 200 rpm) and the liquid was removed. The particles were dried

down in the vacuum concentrator.

After this step, gel pieces could be stored at – 20 ° C until the in-gel digestion.

2.2.11.2.1.4 In-gel digestion with trypsin After washing, dried gel pieces were rehydrated at 4 ° C in 20 µl of pre-cooled

digestion buffer containing 12.5 ng Trypsin per µl in 50 mM NH4HCO3. After 15 min

additional buffer was added, in case all solution had been absorbed, so that the

pieces were all covered with liquid. After 30 to 45 min of incubation the remaining

Trypsin solution was removed. The gel pieces were briefly washed with 80 µl 50 mM

NH4HCO3 to remove the trypsin outside the gel pieces. 80 µl 50 mM NH4HCO3 were

added to keep the gel pieces wet during digestion, which was performed for 16 – 20

h at 37° C. Condensed water droplets were then spun down and the supernatant,

which could already contain small peptides (= Digestion solution), was transferred to

75

a new 96-well plate. After freezing the digestion solution in liquid nitrogen,

evaporation in the vacuum concentrator was performed.

2.2.11.2.1.5 Extraction of peptides from the gel pieces After in-gel digestion and removing of the digestion solution peptides were extracted

from the gel pieces as follows:

Twenty µl 25 mM NH4HCO3 were added to the gel pieces and the liquid was pipetted

3 times up and down. After 1 min centrifugation the samples were incubated for 15

min at 37° C. ~ 80 µl acetonitrile (two times the volume of particles) were added and

the liquid was pipetted 3 times up and down. Gel spots were incubated for 15 min at

37° C and sonicated for 5 min in the sonication bath at 37° C. The liquid (= Basic

extracted peptides) was spun down and transferred to the corresponding wells of the

96 well plate containing the already (partially) evaporated digestion solution. The

pooled solutions were freezed in liquid nitrogen and submitted to further evaporation

in vacuum concentrator. 40 -50 µl of 5 % (v/v) formic acid were added to the gel

pieces and pipetted 3 times up and own. After incubation for 15 min at 37° C and

sonication for 5 min at 37° C, the liquid (= Acid extracted peptides) was spun down

and pooled with the digestion solution and the basic extracted peptides. The pooled

extraction solutions were freezed in liquid nitrogen and dried completely in the

vacuum concentrator over night. The dried samples samples were stored at -20° C

until analysed in mass spectrometry.

76

2.2.11.2.2 Sample preparation starting from streptavidin sepharose affinity capture

A gel-free approach for the tryptic digestion of proteins (see Fig. 2.14) was carried

out in addition to the above mentioned in-gel digestion. This approach will be outlined

in the following sections:

Tryptic „on-resin“ digestion

(2.2.11.2.2.2)

Peptide analysis by µLC-MS/MS

(2.2.11.3)0

20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00


(2.2.11.2.2.1)

Protein extraction(2.2.5)

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Peptide purification(2.2.11.2.2.3)

Tryptic „on-resin“ digestion

(2.2.11.2.2.2)

Peptide analysis by µLC-MS/MS

(2.2.11.3)0

20

40

60

80

10045.23

42.5234.0238.1033.59 49.94

17.05 32.58 54.3526.1114.68 19.11 60.6361.91 71.000.564.00


(2.2.11.2.2.1)

Protein extraction(2.2.5)

Biotin

Biotin

wash

Biotin

Biotin

wash

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Biotin

Peptide purification(2.2.11.2.2.3)

Fig. 2.14 Schematic overview of the on resin

digestion procedure

77

2.2.11.2.2.1 Affinity capture of biotinylated proteins

Protein extracts from mice in vivo biotinylated either with Sulfo-NHS-LC-Biotin or with

Sulfo-NHS-SS-biotin were submitted to affinity capture of the biotinylated protein

according to the protocol specified in 2.8.

2.2.11.2.2.2 On resin digestion The streptavidin sepharose carrying the biotinylated proteins was transferred to a

centrifugal filter device, and centrifuged (1 minute at 16’100 x g). Several washing

steps were performed: The SA sepharose was washed three times with 400 µl buffer

A and two times with 400 µl buffer B. Between the different washing steps the

samples were centrifuged (1 minute, 16’100 x g) and the flow through was discarded,

and the resin was resupended in the next solution by gently vortexing. After washing,

100 µl of 50 mM NH4HCO3 were added to each sample and pipetted up and down

several times to resuspend all the SA sepharose. The SA sepharose suspension was

transferred to a 1.5 ml safe-lock Eppendorf tube and 5 µl of Trypsin stock solution

(concentration: 0.04 µg/µl) were added. The Eppendorf tubes were closed firmly and

were sealed with a piece of parafilm. The sample was vortexed gently. The tryptic

digestion was performed overnight at 37°C on an orbital shaker.

2.2.11.2.2.3 Peptide purification

Fig. 2.15 ZipTips

After tryptic digestion the SA sepharose was resuspended and transferred into a

centrifugal filter device and centrifuged (1 minute, 16’100 x g). The filter with the

78

remaining SA sepharose was discarded. The flow through was dried in the vacuum

concentrator and redissolved in 15 µl 0.1 % TFA (v/v). ZipTips were washed twice

with wetting solution and equilibrated twice with equilibration solution. The sample

was pipetted 20 times up and down with the equilibrated ZipTips to load the peptides

on the tip resin. ZipTips were washed with the washing solution. 10 µl of the elution

solution were transferred in a fresh Eppendorf tube and the peptides were eluted

from the C18 column by pipetting up and down 6 times. The samples were stored at -

20°C.

2.2.11.3 Mass spectrometry

Fig. 2.16 Finnigan LCQDECA ion trap mass spectrometer

Microcapillary liquid chromatography-tandem mass spectrometry (µLC-MS/MS) was

the technique employed for protein identification. Trypsin-digested protein samples

were analyzed employing a Finnigan LCQDeca ion trap mass spectrometer

(ThermoFinnigan) coupled to a Rheos CPS-LC pump (Flux Instruments). Mobile

phases were 0.5% formic acid, 5% acetonitrile (Buffer A) and 0.5% formic acid in

acetonitrile (Buffer D). For the chromatography self-made columns were used.

79

2.2.11.3.1 Preparation of microcapillary liquid chromatography columns

Glass capillaries were pulled with a laser capillary puller. The silicon coating of the

capillary (ID: 75 µm, OD: 375 µm) with an appropriate length (~30 cm) was removed

in the middle of the capillary by heating with a lighter followed by cleaning with

methanol. The capillary was fixed in the puller so that the uncoated piece would be

exposed to the laser beam (heat: 225, velocity: 20, delay: 128). The resulting tips

were carefully opened with a special glass cutter under the microscope.

Columns prepared as described above were filled with C18 beads resuspended in

50% or 100% methanol and transferred to a vial containing a magnetic stir bar. The

vial was placed in a pressure cell. During column filling, the C18 suspension was

continuously mixed with a magnetic stirrer. The cover of the chamber was closed and

fixed. The glass capillary was inserted with the tip pointing up through the valve of the

pressure cell and the valve was closed. The cell was set under pressure (20-30 bar

Helium) and 10 cm of the capillary were filled with beads. The freshly prepared

column was then washed with buffer A for at least 10 min.

2.2.11.3.2 Standard operating procedure to check performance After installation of a new column 50 fmol of tryptic digests of cytochrom C and

human serum albumin were submitted to µLC-MS/MS analysis, performed as

described below for the sample measurements. A successful identification of these

standard proteins indicated a satisfying performance of the column and the proper

operation of the system.

2.2.11.3.3 Sample analysis All steps were performed in a sterile environment to prevent the sample from

contamination. The frozen peptides were dissolved in 10 µl of buffer A and vortexed

80

briefly before centrifugation. The samples were transferred from the 96 well plate to a

PCR tubes and submitted to LC-MS/MS analysis as follows.

With a 25 µl syringe 6µl sample were loaded onto the C18 column using the PAL

autosampler. The sample was injected into a 5 µl sample loop which was cleaned

afterwards four times with 25 µl buffer A. The syringe was pre-cleaned five-times and

post-cleaned four times with 25 µl of buffer A.

Peptides were eluted applying a gradient of 5 - 90 % (v/v) acetonitrile in water in the

presence of 0.5 % (v/v) formic acid.

Spectra were acquired for 65 min in an automated MS/MS mode, consisting of four

sequential events: a full scan mass spectrum was followed by three tandem mass

spectra scans on the most intense, the second most intense and the third most

intense ion detected in the full scan mass spectrum. These four scan events were

repeated throughout the LC run. A dynamic exclusion of ions measured in any

MS/MS scan was automatically carried out for 30 second.

2.2.11.4 Data evaluation If a peak in a MS/MS spectrum showed a total ion current (=TIC) intensity of more

than 5.0 x 104, it produced one DTA-file. Normally between 100 and 1000 DTA-files

were obtained during one µLC-MS/MS run.

2.2.11.4.1 Sequest analysis The uninterpreted MS/MS data were matched against theoretical tryptic digest digest

data for proteins present in a mouse protein database, which was downloaded from

the NCBI homepage using the SEQUEST software (version 2.0).

Generally, a cross correlation factor (Xcorr) higher than 2.5 indicates a highly

significant match, whereas a delta cross-correlation factor (dCn) higher than 0.1

indicates a significant distinction between the best match and the second-best match.

81

82

Additionally, MS/MS spectra were validated manually to assume good quality spectra

with fragment ions clearly above baseline noise and serial matches in the b or y

series.

2.2.12 Synthesis of a dextran-biotin-NHS derivative 2.2.12.1 Buffers and solutions PB (10x) 200 mM NaH2PO4

300 mM Na2HPO4

In MilliQ

PB 20 mM NaH2PO4

30 mM Na2HPO4

In MilliQ

Elution buffer (1) 50 % (v/v) DMSO

50 % (v/v) MilliQ

Elution buffer (2) 50 % (v/v) DMSO

5 % (v/v) PB (10x)

45 % (v/v) MilliQ

Elution buffer (3) 100 % (v/v) PBS

Elution buffer (4) 10 % (v/v) PB (10x)

90 % (v/v) MilliQ

Elution buffer (5) 7.5 % PB (10x)

67.5 % MilliQ

25 % DMSO

83

Elution buffer (6) 10% PB (10x)

90% MilliQ

2.2.12.2 Synthesis A commercially available 70 kDa dextran modified with 5 lysins and 40 biotins was

reacted with a 100 x excess of disuccinimidyl tartrate (DST), which acts as

crosslinker of primary amino groups (see Fig. 2.17). The excess of crosslinker was

used in order to reach the reaction of each dextran lysine residue with another DST

molecule, avoiding crosslinking of amino groups of the same or of different dextran

molecules.

100-fold excess

70 kDa dextranmodified

with 5 biotinsand 40 lysins

Dextran-biotin-NHS-derivative

Crosslinker DST featuring 2 amino-

reactive NHS-groups

100-fold excess

70 kDa dextranmodified

with 5 biotinsand 40 lysins

Dextran-biotin-NHS-derivative

Crosslinker DST featuring 2 amino-

reactive NHS-groups

Fig. 2.17 Scheme of the synthesis reaction

2.2.12.2.1 Labelling of the modified dextran 12.5 µl of 2 mM Fluorescein-5-isothiocyanate in DMSO, 250 µl 40 µM modified

Dextran in MilliQ and 237.5 µl DMSO were gently vortexed and incubated for 2 hours

at room temperature. A 25 µM labelled, modified Dextran stock resulted.

84

Biotin

Fluorescein-5-thioisocyanat

Lysin

Basic element of the dextran polymer

Biotin

Fluorescein-5-thioisocyanat

Lysin

Basic element of the dextran polymer

Fig. 2.18 Labelled modified dextran

2.2.12.2.2 Linking of the modified, labelled Dextran with Disuccinimidyle Tartrate (crosslinker)

625 µl 40 mM Disuccinimidyle Tartrate (DST) in DMSO and 250 µl labelled, modified

Dextran in 50 % (v/v) DMSO solution (established as described above) were

vortexed gently and incubated for 15 minutes at room temperature.

2.2.12.3 Purification 2.2.12.3.1 Spin dialysis After applying the sample to the sample reservoir, the first centrifugation step was

performed for 5 min at 14’000 × g and the concentrate was diluted to 500 µl.

Centrifugation and dilution were repeated four more times. Each flow through and the

final diluted concentrate were collected and the absorption at 490 nm was measured

with a plate reader. The results were plotted in a bar chart.

2.2.12.3.2 Gelfitration

A pre-packed PD-10 Desalting column containing Sephadex G25 medium was used

for gel filtration. The PD-10 column was equilibrated with 25 ml elution buffer. The

sample was added to the column and completed to 2.5 ml with elution buffer. 0.5 ml

85

fractions of the flow-through were collected with Eppendorf tubes. The elution was

performed with up to 30 ml of elution buffer and 0.5 ml fractions of the flow-through

were collected.

Fig. 2.19 PD-10

desalting columns

2.2.12.3.3 Evaluation of the purification by absorption measurement

200 µl of each fraction were transferred to a 96 well-plate and the absorption was

measured with a VERSAmax Microplate Reader at 490 nm. The measurement was

controlled by SOFTmax PRO software. The absorption values were evaluated by

using Excel as software.

2.2.12.3 Activity test The reaction mixture of the modified, labelled Dextran-biotin-NHS-derivative was

submitted to a PD-10. 5 µl of Fluoresceinamin stock were added to the fractions 8, 9,

10, 11 and 12. The reaction mixture was vortexed gently and was incubated for at

least 15 minutes at room temperature. Another PD-10 was performed, the absorption

at 490 nm of the fractions was measured with the ELISA plate reader and the

absorption values were evaluated by using Excel as software.

86

3 RESULTS 3.1 Optimization of the in vivo biotinylation of tumours In vivo biotinylation of tumour-bearing mice had already been routinely performed by

Rybak et al. in our laboratory. However, only a minor part of the perfused tumours

were successfully biotinylated according to histochemical staining experiments and

streptavidin blot analyses of protein extracts, whereas normal organs seemed to be

easily accessible for in vivo biotinylation (unpublished data obtained by Rybak et al.):

Heart, kidney, liver, muscle, and tongue of almost every in vivo biotinylated mouse

showed positive staining, while in negative controls (perfusion with saline) only a

slight background staining in certain tubular structures of the kidney was visible

(probably due to the presence of biotin-carrying carboxylases). In in vivo biotinylated

mice the strongest staining was found in kidneys. While the medulla (data not shown)

was moderately stained, tubular structures in the cortex and especially the glomeruli

showed strong staining. The liver was mainly stained around vascular structures,

however, the biotinylation reagent had entered the liver parenchyma to a certain

extent which varied within different parts of a liver as well as within different mice. In

muscle tissue strong staining was found around vessel structures as well as in the

intercellular spaces between the muscle fibers, where also the endomysial capillaries

are located, whereas the inner parts of the muscle fibers were not stained.

Also a number of tumours could be biotinylated successfully, which in the

histochemistry experiments usually resulted in the staining of structures which

correspond to blood vessels and of certain particles which could represent apoptotic

or necrotic cells. However, the perfusion of tumours was much less efficient and

more heterogeneous compared to other organs. While some tumours showed a

homogenous staining of vessel-near structures all over a cross-section, often

tumours were only stained in a part of the section and not stained at all in other parts.

In other tumours only some few vessels were stained within the section or just small

parts, often at edges, were stained which probably represented artifacts.

Furthermore, a co-staining of tumour sections from in vivo biotinylated mice against

biotin and the endothelial marker CD31 on the one hand revealed that biotinylated

87

sites corresponded well to CD31-positive structures, but on the other hand showed

that there are parts of the tumour with positive CD31 staining which are not

biotinylated. Overall, with the original perfusion protocol only 7.5% of the tumours

could be biotinylated successfully at least in a significant part of the cross-section

(see Fig. 3.3). Also the application of Heparin to prevent blood clogging did not

improve the in vivo biotinylation of tumours (Rybak et al., unpublished).

Thus, we set out to improve the perfusion of tumours altering the perfusion protocol.

3.1.1 Perfusion with a higher flow rate In the original perfusion protocol applied so far, the perfusion needle consisted of a

modified 25 G butterfly cannula connected to small diameter tube (0.5 mm ID), which

led to a perfusion flow rate of about 1.5 ml/min. However, the heart minute volume of

a mouse, that means the rate with which the heart pumps the blood through the

circulation in the living animal, is 11-36 ml/min (!) [Poole T.B., 1999]. Thus, it was

hypothesized that the incomplete perfusion of tumours could be due to a too low

perfusion flow rate. An optimization attempt featuring higher perfusion flow rates was

attempted.

F9 tumour-bearing SvEv/129 mice were submitted to in vivo biotinylation according to

the procedure described in 2.5 using the following perfusion conditions:

Perfusion

needle Perfusion pressure

Perfusion flow rate

Temperature of perfusion

solutions

Warming the

animal

1. Perfusion

step: washing

2. Perfusion step:

biotinylation

3. Perfusion

step: quenching

Original protocol

25G butterfly

with small

diameter tube

100 mm Hg

~1.5 ml/min

RT No 10 min PBS

10 ml Sulfo-NHS-LC-biotin 1 mg/ml in PBS

10 min 50 mM

Tris in PBS

Tabl. 3.1 Perfusion conditions (original protocol)

88

In a series of experiments, mice were submitted to an in vivo biotinylation procedure

(see 2.2.2) with Sulfo-NHS-LC-Biotin applying either the original protocol (25 G

cannula equipped with a tube of 0.5 mm inner diameter) with a flow rate of ~1.5 ml/

min or a modified protocol. In the modified version, cannulae with the same or a

larger diameter (provided with a barb by Martin Mörter, see Materials) and tube with

not limiting diameter was used, leading to higher perfusion flow rates, and higher

volumes of biotinylation solution were applied (see table 3.2).

Protocol variant

Size of perfusion cannula

limiting 0.5 mm ID tube

resulting perfusion flow

rate

Volume of perfusion solution

resulting time for perfusion with

biotinylation solution1 25 G yes ~1.5 ml/min 10 ml ~7.0 min

2 25 G no ~4 ml/min 10 ml ~2.5 min

3 23 G no ~8-10 ml/min 40 ml ~8-10 ml/min

4 23 G no ~5 ml/min 80 ml ~16 min

5 21 G no ~18-20 ml/min 80 ml ~4-5 min

6 21 G no ~20 ml/min 160 ml ~8 min

Tabl. 3.2 Various perfusion conditions with a higher flow rate

After perfusion, the organs of interest (liver, kidney, muscle and tumour) were

excised, embedded in cryomedium and snap-frozen. Section from heart, liver, kidney,

muscle and tumour were cut and submitted to histochemical analysis with

Streptavidin:biotinylated alkaline phosphatase/FAST-Red TR (red staining of

biotinylated structures) and counterstaining with Haematoxylin (blue staining)

according to the protocol specified in 2.2.4 (data only shown for kidney, liver, muscle

and tumour of mice perfused according to protocol variant 1 and 6, see Fig. 3.1)

Overall, the histochemical analysis revealed that no significant improvement of

tumour perfusion could be achieved by applying higher perfusion flow rates. In heart,

kidney and liver, more intense staining of biotinylated structures was observed and

biotinylation was present not only mainly around vessel structure but also deeper in

the tissue compared to the original protocol. However, besides of the tumours, also

89

the muscle sections did not exhibit positive staining, suggesting that also this tissue is

not perfused when applied the variant protocols.

Taken together with the observations that, during the perfusion experiments with high

flow, the perfusate often flew out of the nose and the mouth of the animal and that

the abdomen and specially the intestine of the mice swelled, our obsevations indicate

that vessels might be disrupted during the perfusion with the high flow rates.

Figure 3.1 shows the histochemical analysis of liver, kidney, muscle, and tumour

sections derived from mice perfused either according to protocol variant 6 (see table

3.1 and figure 3.1 A) or according to the original protocol (protocol variant 1 in table

3.2, see figure 3.2 B).

Liver Kidney Muscle Tumor

A

B

Liver Kidney Muscle Tumor

A

BFig. 3.1 Histochemical detection of biotinylated structures of organ sections from mice perfused with sulfo-NHS-LC-biotin A: Perfusion with high flow rate (protocol variant 6 in Tabl.

3.2) B: Perfusion according to the original protocol (protocol

variant 1 in Tabl. 3.2)

90

3.1.2 Perfusion versus intravenous injection of mice with Hoechst dye

In the living animal, the blood should be able to reach the tumour as it needs supply

of oxygen and nutrients to grow. In order to investigate why the tumour is perfused in

a living mouse, but difficult to access in the artificial perfusion procedure, we

compared the accessibility of vascular structures for Bisbenzimide H33342 (Hoechst

dye), a nuclei-staining fluorescent dye, either by intravenous injection or by perfusion.

Liver KidneyHeart TumourMuscle Exposure time: 500 µsec

Intravenous injection of

Hoechst dye

Hoechst dye perfusion

with previous

saline wash

(1000 µsec)

Hoechst dye perfusion without

previous wash

(2000 µsec)

Fig 3.2 Sections of heart, liver kidney and muscle derived from mice either perfused or injected intravenously with Hoechst dye (See text for explanation.)

91

Mice received an intravenous injection of 100 µl 1 mg/ml Bisbenzimide H 33342

(Sigma) in an aqueous solution of 0.9% (w/v) NaCl. In a simplified calculation and

with assume a blood volume of 2 ml, this injection corresponded to a total

concentration of 0.05 mg/ml in the blood. After 2 or 4 min the animal was sacrificed.

Other mice were anaesthetized, underwent surgery as described in 2.2.2 and were

perfused with 0.05 mg/ml Hoechst dye in an aqueous solution of 0.9% (w/v) NaCl for

4 min. A washing perfusion step with 0.9% NaCl solution was either performed for 10

min prior or for 6 min after the perfusion with the fluorescent dye to test the impact of

the previous wash step. Immediately after sacrificing the injected animals or after

perfusion respectively, organs were excised, embedded in cryoembedding compound

and snap-fozen. Sections were cut and analyzed using a fluorescence microscope as

described in 2.2.4.

The microscopic analysis of organ sections from mice injected intravenously with the

dye (Fig. 3.2, upper panels) revealed that normal organs as heart, liver, kidney, and

muscle showed a homogenous staining of nuclei all over the tissue, whereas tumours

were only stained around blood vessels. This indicates, that in the living animal the

blood perfusion of tumours is worse than of normal organs, but at least present to a

certain extent. However, experimental perfusion with a similar final concentration of

the same dye for a similar time frame after having washed away blood and cells with

PBS for 10 min (see Fig. 3.2, middle panels) showed, that the tumour seemed not to

be reached by the dye. Furthermore, under these conditions, also in heart and liver

only structures near larger blood vessels, and in kidney only certain vessels and

glomeruli were stained, whereas in most parts of these tissues staining was absent.

Performing the perfusion with Hoechst dye prior to the saline wash (see Fig. 3.2,

lower panels) seemed to improve the accessibility of microvessels within these

tissues, because in this case they exhibited a staining more similar to the in vivo

situation. However, staining of larger vessels and of the glomeruli in the kidney still

seemed to be stronger than in the rest of the tissue. Notably, also in these optimized

conditions, solid tumour comtinued not to show a specific staining.

These results gave evidence that the PBS washing steps prior to perfusion with the

active reagent might reduce the accessibility of the vessels. For the in vivo

biotinylation procedure, these findings prompted us to omit the washing step prior to

the perfusion with biotinylation reagent.

92

3.1.3 In vivo biotinylation with an optimised protocol Before testing the omission of the PBS wash step (see 3.1.2) in further in vivo

biotinylation experiments, we took two other aspects into account, which led us to an

final optimized perfusion protocol:

1) We reasoned that the low accessibility of tumours for the in vivo biotinylation

might also be a result of a collapse of microvessels due to a too low oncotic

pressure of the perfusion solutions. An influx of water from the perfusion

solution into the tissue might occur leading to a compression of the

microvessels. Therefore we decided to supplement the perfusion solutions

with dextran 40. This ~40 kDa polymer binds water molecules and therefore

increases the colloidal osmotic (oncotic) pressure. In the form of an isotonic

10% (w/v) solution in saline it is used clinically as plasma expander.

2) The reduced body temperature of the mouse in anesthesia and during the

perfusion might lead to a circulatory shock of the mouse. In a physiological,

unconscious self-protection mechanism, the cool environment as well as the

injury during surgery possibly leads to a vasoconstriction in the periphery of

the body aiming at the maintenance of the function of the more important inner

organs. By warming the the perfusion solutions to the body temperature of the

mouse (~38°C) and keeping the animal warm in anesthesia as well as during

surgery and perfusion, we aimed at the better perfusion of tumours.

Thus, several in vivo biotinylation experiments were performed with modified

protocols. Overall, 21 tumour-bearing mice were submitted to in vivo biotinylation

applying the original protocol but without the previous PBS wash and with 10%

dextran 40 in the biotinylation and the quenching solutions (see Tab. 2.1). Additional

7 mice were perfused according to the final optimized in vivo biotinylation protocol

specified in 2.2.2 (see Tab. 2.1) featuring the omission of the PBS wash prior to the

biotinylation step, the pre-warming of the perfusion solutions to 38°C, the warming of

the animal during anesthesia and perfusion using a heating pad and an infrared

lamp, as well as the presence of 10 % (w/v) Dextran 40 in the perfusion solutions.

93

Using dextran 40 in the perfusion solutions, the flow rate was decreased due to the

higher viscosity. Thus the 25 G perfusion needle was used without the limiting low

diameter tube which was part of the original protocol. By this, a perfusion flow rate of

~1.8 ml/min was applied.

From organs and tumours cryosections were prepared and submitted to

histochemical analysis for the detection of biotinylated structures as described before

(for protocols see 2.2.4 (individual data not shown)).

The influences of these modifications were analysed by a statistical evaluation of the

histochemical results of these experiments in comparison to previous experiments

performed by Rybak et al. using the original perfusion protocol. In this analysis (Fig.

3.3), tumours perfused so far (n=42 for the original protocol (Fig. 3.3 column II); n=21

for the perfsuion without PBS wash and with dextran in the perfusion solutions (Fig.

3.3, column III); n=7 for the final optimised protocol (Fig. 3.3, column IV) were

assigned to the following groups (Fig. 3.3 column I):

A) tumours which did not show any staining of biotinylated structures in the

histochemical analysis

B) tumours, in which only certain structures at the edges were positively

stained for biotin (this includes some cases in which apparent vascular

structures were stained near the edge and other cases in which only the

outer ring of the tumour was stained which may be an artefact)

C) tumours in which a significant part of the tumour (varied between ~10-70%

of the section area) was showing positive staining of apparent vascular

structures

D) tumours in which more or less all of the section (80-100%) exhibited

positive staining of apparent vascular structures

94

5%

62%

31%

2.5%

(n=42)

5%

19%

57%

19%

(n=21)

Modified:- no PBS wash- with dextran

Original protocol

Schematicview of the

tumor section

14%

43%

14%

29%

(n=7)

I II III IV

A

B

C

D

Optimized:- no PBS wash- with dextranwith warming

Fig. 3.3 Preliminary comparative statistic of the effects of addition of plasma expander to the perfusion solutions and omission of the previous washing step (III) as well as of the warming of the animal and the perfusion solutions (IV) in the in vivo biotinylation procedure. See text for detailed explanations.

This comparison (Fig. 3.3) showed that, while only 7.5% of the tumours perfused

according to the original protocol were biotinylated at least in significant part

(categories C + D), the proportion increased to 24% of the tumours perfused without

the PBS washing step and with dextran in the perfusion solutions, and even to 43%

of the tumours perfused according to the final optimised protocol including omission

of the PBS wash, addition of dextran to the perfusion solutions, pre-warming of the

perfusion solutions to 38°C and warming of the animal during anesthesia and

perfusion.

These results indicate that the increase of the colloidal osmotic pressure improved

the accessibility of vascular structures in the tumour during the perfusion procedure.

Even though the number of animals submitted to the final optimized in vivo

95

96

biotinylation protocol (n=7) is too low to draw definite conclusions, the results suggest

that the use of pre-warmed perfusion solutions and the warming of the animal during

anesthesia and perfusion further improves the perfusion of tumours.

3.2 Gel-based proteomic analysis After in vivo biotinylation biotinylated proteins were purified on streptavidin sepharose

and submitted to a gel-based proteomic analysis for the identification of accessible

proteins in the different tissues.

In vivo biotinylation experiments were performed according to the procedure

described in 2.2.2. The protocol included an initial PBS wash followed by the

biotinylation with 10 ml sulfo-NHS-LC-biotin (1 mg/ml) in PBS during 7 minutes and

the quenching of excessive biotinylation reagent by a 10 min washing step with a 50

mM Tris in PBS. Histochemical staining of organ sections with streptavidin indicated

successful biotinylation of vascular structures (data not shown). As negative controls

samples were used, which were prepared from a mouse, which underwent the

analogue procedure in which, however, the biotinylation solution was replaced by

pure PBS.

3.2.1 Purification of biotinylated proteins on streptavidin Biotinylated proteins were purified from different organs as follows: Organ specimens

were homogenized in 20 µl lysis buffer per mg tissue according to the protocol

specified in 2.2.5.1, total protein extracts were prepared as described in 2.2.5.2 and

the protein concentration was determined. The protein extracts were diluted in lysis

buffer and, for each of them, 2.08 mg protein in 500 µl (IN) were added to 100 µl

streptavidin sepharose resin which had been previously washed. Washing and

capturing was carried out as described in 2.2.6, but the capturing was performed at

RT to avoid precipitation of SDS. For elution of biotinylated proteins, 500 µl release

solution were added followed by incubation for 15 min at RT, followed by incubation

for 15 min at 96°C. Afterwards, the resin was pelleted by centrifugation for 5 min at

16’100 x g and the resulting supernatant was collected (REL). For analysis of the

purification, 24 µl of each extract before (IN) (corresponding to 100 µg protein) and

supernatant after (SUP) incubation with streptavidin sepharose as well as the

biotinylated proteins after release (REL) from the resin were subjected to SDS-PAGE

(upper panels) which was carried out as described in 2.2.9. Gels were stained with

SYPRO RUBY. In addition, proteins were blotted onto a nitrocellulose membrane and

97

biotinylated proteins were detected by streptavidin-horseradish peroxidase and the

ECL+Plus Western blotting detection system.

Figure 3.4 shows the SDS-PAGE and SA blot analysis of biotinylated proteins,

recovered from homogenates of muscle, liver and kidney, from mice which had been

perfused with sulfo-NHS-LC-biotin (+) or saline (-).

In the input and in the supernatant of the Sypro Ruby stained gels a lot of intense

bands were detectable, whereas only few weak bands were present in the elute

(REL). This indicates that only few proteins were biotinylated and, thus, captured on

and eluted from the SA sepharose.

In the SA blot, which can only detect biotinylated proteins, several biotinylated

proteins were detectable in the input and in the eluted fractions.

The absence of biotinylated bands in the “SUP” lines of the streptavidin blots

indicates that the capture of biotinylated protein was quantitative. The fact that the

eluate essentially exhibits the same band pattern as the INPUT indicated that the

purification yielded a quantitative recovery of biotinylated proteins.

It should be noted that the pattern of proteins detected in the “REL” fractions of the

streptavidin blot does not overlap completely to the one observed in the “IN” fraction,

in particular in the low-molecular-weight region. This may be explained at least in part

with the different buffers (lysis buffer or elution buffer) in which the proteins are

present. Furthermore, the presence of an excess of non-biotinylated proteins in the

“IN” fractions could partially mask the less abundant, biotinylated proteins in the

same fraction. Finally, a certain degree of protein degradation cannot be ruled out at

this stage.

Muscle, liver and kidney extracts from mice perfused only with PBS (negative

controls, “-“) exhibited only a few biotinylated bands. These are enzymes (such as

pyruvate carboxylase) which use biotin as cofactor.

98

M+

IN SUP

REL

M-

IN SUP

RE L

L+

IN SUP

RE L

L-

IN SUP

REL

K+

IN SUP

REL

K-

IN SUP

RE L

25015010075

503725201510

SDS-PAGE

Streptavidin blot

M+

IN SUP

REL

M-

IN SUP

RE L

L+

IN SUP

RE L

L-

IN SUP

REL

K+

IN SUP

REL

K-

IN SUP

RE L

25015010075

503725201510

SDS-PAGE

Streptavidin blot

kDa

Fig 3.4 SDS-PAGE and SA blot analysis of biotinylated proteins, recovered from homogenates of muscle (M), liver (L) and kidney (K), from mice which had perfused with sulfo-NHS-LC-biotin (+) or PBS (-) IN: protein extract diluted with lysis buffer containing 2.08

mg protein / 500 µl SUP: supernatant collected after incubation with SA sepharoseREL: supernatant collected after elution of the biotinylated

proteins from the SA sepharose

In summary, the results indicate a successful purification and quantitative recovery of

in vivo biotinylated proteins. The recovered proteins were subsequently submitted to

protein identification by mass spectrometry analysis.

3.2.2 Excision of gel bands and protein identification by LC-MS/MS

The eluted biotinylated proteins (REL) obtained as described in 3.2.1 were submitted

to a further SDS-PAGE applying more protein on the gel to increase the starting

material for a subsequent mass spectrometry analysis of protein bands.

99

28 µl (maximal loading volume) of the eluted fraction (REL) (corresponding to 118 µg

protein in the original extract “IN”) was loaded on the gel. The gel was stained with

Sypro Ruby stain and the visible bands were excised as described in 2.2.10. The

proteins were reduced and alkylated (see 2.2.11) and submitted to an in-gel digestion

with trypsin (see 2.2.11). After extraction from the gel pieces the resulting peptides

were submitted to protein identification using liquid chromatography-tandem mass

spectrometry analysis according to the procedure specified in 2.2.10.

The following proteins could be identified: (Fig 3.5 C) albumin (perhaps indicating

incomplete removal of blood components during perfusion), (Fig. 3.5 D) streptavidin

fragments (resulting from the capture and release procedure), (Fig. 3.5 A) pyruvate

carboxylase as well as (Fig. 3.5 F) propionyl CoA-carboxylase (both are enzymes

carrying physiologically biotin as a prosthetic group) and, a protein which was found

to be specifically biotinylated during the in vivo biotinylation in the kidney and was

identified as the kidney-specific tubular membrane protein (Fig. 3.5 B) cadherin 16.

Also several weaker bands (G) were analysed but did not lead to reliable

identifications.

In summary, the gel-based proteomic analysis suffer from poor sensitivity, as only

few protein bands were at all visible in the gel and only the most intense bands led to

protein identification.

100

250

150

100

75

50

37

2520

1510

M+ M- L+ L- K+ K-

Release samples

E

F

HG

G

G

G

G

G

250

150

100

75

50

37

2520

1510

M+ M- L+ L- K+ K-

Release samples

E

F

HG

G

G

G

G

G

kDa

A

B

D

C

A

B

D

C

Fig 3.5 SDS-PAGE analysis of biotinylated proteins, recovered by SA affinity purification from homogenates of muscle (M), liver (L) and kidney (K), from mice which had been perfused with sulfo-NHS-LC-biotin (+) or PBS (-). The bands of the biotinylated proteins were excised and submitted to tryptic in-gel digestion followed by mass spectrometric analysis. The criteria mentioned in 2.2.11.3.1 were applied to validate the data obtained from the SEQUEST analysis. Identification (A-F) provided with a question mark did not completely fulfill the criteria. A Pyruvate carboxylase

and Annexin? B Kidney-specific Cadherin 16 (90kDa, one type

membrane protein) C Albumin (66 kDa) D Streptavidin fragments (resulting from the

capture and release procedure) E Proprionyl CoA-carboxylase ? F Protocadherin 8 ? (10kDa)

Ras related protein Rab33A ? (brain and ovarian, 27 kDa) Mediator of RNA-polymerase II transcript subunit 8 homolog ? (nuclear)

G Not identified H Technical problems

101

3.2.3 Optimization approach I: Protein concentration on resin

To improve the sensitivity of the gel-based proteomic analysis (see 3.2.2), we aimed

at loading a higher protein amount on the gel. However, the loading volume is limited,

therefore forcing us to try and increase the protein concentration in the eluate of the

affinity purification.

In a first attempt, we tried to reach a higher concentration of purified biotinylated

proteins by applying more protein, but on less streptavidin sepharose allowing the

elution in less elution buffer, thereby having finally more eluted proteins in a smaller

volume.

By this concentration on the resin, we aimed at achieve an 8-fold increase of protein

concentration, using the same protocol as described above (see 3.2.1 and 2.2.6), but

including the following changes:

1. 1000µl instead of 500µl of protein extract (protein concentration:4.2 µg protein

/ µl) were added to the Streptavidin Sepharose.

2. Less SA sepharose slurry (160 µl instead of 320 µl) was applied in the

capturing procedure

3. Less elution buffer (150 µl instead of 500 µl) was added to release the

biotinylated proteins from the SA sepharose

Applying this modified protocol, tissue extracts (liver, muscle, kidney and tumor; data

for liver and muscle not shown) from three different tumour-bearing mice were

purified. One mouse was perfused only with PBS as negative control (-). The two

other mice were biotinylated in vivo applying similar protocols; however, one of these

mice (++) exhibited strong biotinylation of vascular structures in all organs and in the

tumor according to histochemical analysis (data not shown), whereas the other

mouse (+) showed only weak biotinylation in histochemistry.

As described in the previous analysis (see 3.2.1), 24 µl of each extract before (IN)

(corresponding to 100 µg protein) and supernatant after (SUP) incubation with

streptavidin sepharose as well as the biotinylated proteins after release (REL) from

the resin were subjected to SDS-PAGE (Fig. 3.6, upper panels) and to streptavidin

blot analysis (Fig 3.6, lower panels).

102

In contrast to the analysis in 3.2.1, the streptavidin blot revealed also biotinylated

proteins in the supernatant, indicating a too low capacity of the streptavidin

sepharose in respect to the protein amount. This is especially true for the kidney

extract, which in general contained much more biotinylated proteins than the tumor

extract, a fact which might be due to the worse perfusion of the tumor (see 3.1).

In the REL lanes, not more protein bands could be detected in comparison to original

purification procedure (see 3.2.1 and 3.2.2) and present bands did not show a higher

intensity. Thus, the aim of this optimisation attempt was not achieved.

Even though biotinylated proteins were recovered from tumors of in vivo biotinylated

mice here for the first time, we did not submit these samples to LC-MS/MS analysis,

because the protein bands in the eluted tumor sample were too faint except of one

band which exhibited the molecular weight of serum albumin.

103

SA blot

SDS-PAGE

T-

IN SUP

REL

T++

IN SUP

RE L

T+

IN SUP

REL

K-

IN SUP

RE L

K++

IN SUP

REL

K+

IN SUP

RE L

25015010075

503725201510

m

1601057550

3530

1525

10

SA blot

SDS-PAGE

T-

IN SUP

REL

T++

IN SUP

RE L

T+

IN SUP

REL

K-

IN SUP

RE L

K++

IN SUP

REL

K+

IN SUP

RE L

25015010075

503725201510

m

1601057550

3530

1525

10

kDa

Fig. 3.6 SDS-PAGE and SA blot analysis of biotinylated proteins, recovered with the changed SA affinity protocol from homogenates of tumor (T) and kidney (K), from mice, which had been perfused with sulfo-NHS-LC-biotin (+ / ++) or PBS (-).For the purification on streptavidin sepharose. ++: mice exhibited strong biotinylation of vascular structures in

histochemical analysis +: mice exhibited weak biotinylation of vascular structures in

histochemical analysis IN: protein extract diluted with lysis buffer containing 2.08 mg

protein / 500 µl SUP: supernatant collected after incubation with SA sepharose REL: supernatant collected after elution of the biotinylated proteins

from the SA sepharose

104

3.2.4 Optimization approach II: Sample concentration by spin dialysis

In the second attempt, we aimed again at the loading of higher protein amount on the

gel in order to improve the sensitivity of a higher protein amount on the gel. Instead of

a protein concentration on resin (first attempt), the supernatant collected after having

released the biotinylated proteins from the resin was submitted to a concentration

procedure by spin dialysis with Microcon centrifugal filter devices (MWCO: 3 kDA or

10 kDa).

A liver protein extract was recovered from a liver homogenate from a mouse perfused

with sulfo-NHS-LC-biotin. The protein extract was diluted in lysis buffer and 4.16 mg

protein in 1000 µl (IN) were added to 640 µl SA sepharose which had been

previously washed according to the protocol described in 2.x. The biotinylated

proteins were eluted by applying 1000 µl elution solution. This purification procedure

was repeated another three times.

500 µl of the supernatant (REL) (containing the biotinylated proteins) after release

from the resin were subjected to each spin dialysis procedure (see 2.x) performed by

using different protocols and different centrifugal filter devices (MWCO: 3 kDa and 10

kDa) (see table below):

Protocol 1 2 3

Spin columns (MWCO: 10 kDa)

Centrifugation for 2 h at 14’000 x g



Spin columns (MWCO: 3 kDa)



Centrifugation for 4 h at 14’000g

After centrifugation, in the approaches centrifuged for 3 and 4 hours precipitates

were present which were dissolved either in 20µl 2% SDS in PBS or in 500µl 2 %

SDS in PBS depending to the protocol (see Fig. 3.7) and centrifuged again for 2.5

hours at 14’000g. In all 6 approaches an approximately 5 fold enhancement of the

protein concentration was achieved.

105

RELREL

Microcon YM-10 and YM-3

Centrifucation: 2 h at 14‘000 x g

=> Precipitation!

+ 500 µl 2% SDS in PBS;

Centrifugation:2.5 h at14000 x g

+ 20 µl 2 %SDS in PBS

~5x concentration

=> Precipitation!

Centrifucation: 3h at 14‘000 x g


~5x concentration ~5x concentration

RELREL

Microcon YM-10 and YM-3

Centrifucation: 2 h at 14‘000 x g

=> Precipitation!

+ 500 µl 2% SDS in PBS;

Centrifugation:2.5 h at14000 x g

+ 20 µl 2 %SDS in PBS

~5x concentration

=> Precipitation!



~5x concentration ~5x concentration

Fig. 3.7 Overview of the different concentration approaches by spin dialysis

For analysis of purification and concentration, 24 µl of each extract before (IN)

(corresponding to 100 µg protein) and supernatant after (SUP) incubation with

streptavidin sepharose and the biotinylated proteins after release (REL) from the

resin as well as the various concentrates were subjected to SDS-PAGE and SA blot

analysis which was carried out as described in 2.2.9. The gel was stained with

SYPRO RUBY.

Fig. 3.8 shows the SDS-PAGE and the SA blot analysis of biotinylated proteins,

recovered from a liver homogenate from a mouse, which had been perfused with

Sulfo-NHS-LC-biotin.

As expected from previous result, in the input (IN) and in the supernatant (SUP) of

the SYPRO RUBY stained gels a lot of intense bands were detectable, whereas only

few weak bands were present in the release (REL). This indicates that only few

protein were biotinylated, thus captured on and released from the SA sepharose.

The lanes of the various concentrated releases exhibit very intense bands in contrast

to the lane of the previous release, indicating a successful concentration. In all REL

106

lanes, there were predominantly the same band pattern detectable showed that no

detectable loss of protein occurred during the concentration by spin dialysis. The

lanes of the concentrated releases exhibit the same signal pattern with similar

intensities indicating that the different concentration approaches led to nearly similar

increases of the protein concentration.

In the SA blot, signals were only detectable in the input (IN) and in the release (REL),

while no bands were detectable in the supernatant (SUP) indicating a successful

purification on SA sepharose. The signal patterns in the REL lane were less intense

than the signal pattern in the INPUT lane indicating a loss of biotinylated proteins

during the purification procedure on SA sepharose. It should be noted that the

pattern of proteins detected in the “REL” fractions of the SA blot does not overlap

completely to the one observed in the “IN” fraction. This fact may be expained as

mentioned in chapter 3.2.1.

The SA blot confirms the conclusion, which was deduced from the SDS-PAGEs: The

lanes of the concentrated releases showed the same signal pattern with the same

intensities indicating that the various concentration approaches led to nearly similar

increases of the protein concentration. The intensity of the signal pattern of the non-

concentrated release was much weaker than the signal patterns of the concentrated

releases, confirming the validity of the various concentration approaches used.

107

REL

3 h

I N S UP

RE L RE L

2 h

REL

4 h

REL

3 h

REL

2 h

RE L

4 h

YM 3 YM 10

SDS-PAGE

250

150

10075503725

20

250__

160__105__

50__35__30__25__15__

75__

10__IN SU

P

REL RE L

2 h

REL

4h

RE L

3 h

RE L

2 h

YM 3 YM 10

REL

4 h

SA blot

REL

3 h

I N S UP

RE L RE L

2 h

REL

4 h

REL

3 h

REL

2 h

RE L

4 h

YM 3 YM 10

SDS-PAGE

250

150

10075503725

20

250__

160__105__

50__35__30__25__15__

75__

10__IN SU

P

REL RE L

2 h

REL

4h

RE L

3 h

RE L

2 h

YM 3 YM 10

REL

4 h

SA blot

kDa

Fig. 3.7 SDS-PAGE and SA blot analysis of biotinylated proteins, recovered from a liver homogenate, from a mouse, which was perfused with sulfo-NHS-LC biotin. IN: protein extract diluted with lysis buffer containing 4.16 mg protein / 1000 µl SUP: supernatant collected after incubation with SA sepharose REL: supernatant collected after elution of the biotinylated proteins from the SA sepharose

REL 2h,3h,4h: supernatant collected after elution and centrifuged for 2, 3 and 4 h processed as described above.

YM3: Spin column (MWCO: 3 kDa) YM10: Spin column (MWCO: 10 kDa)

108

3.3 Fractionation of in vivo biotinylated proteins by hydroxyapatite chromatography for the optimization of the gel-free proteomic analysis

Samples of biotinylated proteins had been analysed previously in our laboratory in a

gel-free proteomic approach by on-resin digestion and shot-gun LC-MS/MS analysis

(see 1.4). A reduction of sample complexity by pre-fractionation of the protein

extracts was attempted, in order to improve mass spectrometry analysis and to lead

to more protein identifications. Therefore, we set up a hydroxyapatite-based

chromatography method for the prefractionation of biotinylated proteins (see 2.2.7),

as this method is compatible with the use of SDS.

3.3.1 Fractionation of a standard protein mixture by hydroxyapatite

chromatography In preliminary experiments, the fractionation of a standard protein mixture containing

Fibrinogen (comprising α, β, and γ-chain), Trypsin, Lysozyme and Pepsin was

optimized to find optimal separation conditions. Figure 3.8 shows the different

working steps of the fractionation of the protein standard mixture. The protein

standard mixture was separated by hydroxyapatite chromatography as described in

2.2.8.

As starting conditions a protocol from Simone Scheurer et al. (unpublished data from

our laboratory) was used:

109

Sample preparation: Filtration of the sample previous to the fractionation Protein amount: 0.96 mg/ml Pepsin 0.48mg/ml Fibrinogen 0.48mg/ml Trypsin 0.48mg/ml Lysozyme Total protein amount: 3 mg Buffer A: 10mM Phosphate, 1% SDS, 0.1mM CaCl2

0.5mM DTE (pH7.2) Buffer B: 500mM Phosphate, 1% SDS, 7.5nM CaCl2,

0.5mM DTE (pH7.2) Fraction size: 1.6 ml Flow rate: 0.8 ml / min Linear gradient of 0 – 100 % buffer B within 30 min

The collected fractions were submitted to SDS-PAGE (2.2.9) with the following

conditions:

• Gels: 12% Bis-Tris, 1mm, 15 wells

• Loading volume: 12 µl

• Protein visualisation: Simply Blue stain

110

Standard

protein mixture Hydroxyapatitechromatography

Different pH(pH 7.2, 6.8, 7.7)

SDS-PAGE of thecollected fractions

Buffer A ( , 1%SDS, 0.1mM CaCl2, 0.5mM DTE)

Buffer B ( , 1%SDS, 7.5 nM CaCl2, 0.5 mM DTE)

Conditions:Fibrinogen (αβγ)TrypsinLysozymePepsin

Standard protein mixture Hydroxyapatite

chromatography

Different pH(pH 7.2, 6.8, 7.7)

SDS-PAGE of thecollected fractions

Buffer A ( , 1%SDS, 0.1mM CaCl2, 0.5mM DTE)

Buffer B ( , 1%SDS, 7.5 nM CaCl2, 0.5 mM DTE)

Conditions:Fibrinogen (αβγ)TrypsinLysozymePepsin

10 mM Phosphate

500 mM Phosphate

10 mM Phosphate

500 mM Phosphate

Fig.3.8 Working steps of the fractionation of the protein standard mixture by

hydroxyapatite chromatography

Fig. 3.9 shows the results of the SDS-PAGE analysis. The three Fibrinogen chains

were not separated from each other in the fractionation by hydroxyapatite

chromatography. All three chains were present together in fractions 15 to 20 with

almost the same band intensity. In each lane of the SDS-PAGE gel, the three chains

were separated as a result of the sample preparation for SDS-PAGE (5 min heating

in presence of β-mercaptoethanol).

Lysozyme was located in several different fractions (F1 to F12) indicating a low

capacity of the column.

Fraction 1 and 2 contained the dead volume (the unbound protein) due to a 2 ml-

loop in front of the detector. The intense bands of several standard proteins

(Fibrinogen, Trypsin and Lysozyme) detected in the first 4 fractions might result from

incomplete initial binding indicating suboptimal retention conditions in buffer A or an

insufficient capacity of the column in respect to the loaded protein amounts. This may

also explain the fact that the lysozyme was spread over so many fractions (F1-F12).

111

250150100755037

25201510

IN mar

F1 F2 F3 F 4 F5 F6 F 7 F8 F 9 F10

F 11

mar

mar

F12

F13

F14

F15

F16

F 17

F 18

F19

F20

F21

F22

Fibrinogen

Trypsin

Pepsin

Lysozyme

250150100755037

25201510

IN mar

F1 F2 F3 F 4 F5 F6 F 7 F8 F 9 F10

F 11

mar

mar

F12

F13

F14

F15

F16

F 17

F 18

F19

F20

F21

F22

Fibrinogen

Trypsin

Pepsin

Lysozyme

kDa

Fig.3.9 SDS-PAGE of a standard protein mixture fractionated by hydroxyapatite chromatography (before optimization) F1 to F12: Fraction 1 to fraction 2 IN: Input: non-fractionated standard protein mixture Mar: Marker

Testing different pH values (6.8, 7.7, data not shown) did not lead to a better protein

separation.

Thus, in a series of experiment (data not shown) various optimization steps were

implemented:

1. An additional sample preparation step was introduced (100mM DTE and 5’

heating) to reduce the disulfide bonds of the Fibrinogen and achieve the

separation of the three chains previous to hydroxyapatite chromatography.

2. Less protein was loaded on the cartridge.

3. The phosphate concentration of buffer A was decreased to achieve the initial

binding of all proteins to the cartridge.

In summary, the experiments with the standard protein mixture led to the following

optimized protocol:

112

Sample preparation: Heating for 5 minutes in presence of

100mM DTE and filtration previous to the fractionation

Protein amount: 0.96 mg/ml Pepsin 0.26 mg/ml Fibrinogen 0.26 mg/ml Trypsin 0.02 mg/ml Lysozym

Total protein amount: 1.6 mg Buffer A: 50mM Tris, 1% SDS, 0.5mM DTE (pH7.2) Buffer B: 500mM Phosphate, 1% SDS, 7.5nM CaCl2,

0.5mM DTE (pH7.2) Fraction size: 1.6 ml Flow rate: 0.8 ml / min Linear gradient of 0 – 100 % buffer B within 30 min

Figure 3.10 shows the SDS-PAGE analysis of the standard protein mixture

fractionated by HA chromatography applying the optimized protocol. The different

proteins were mostly found in different fractions indicating a successful separation.

The elution of each protein was limited to three fractions, which was mainly achieved

by the reduction of the protein amount loaded to the column.

The substitution of the phosphate in buffer A with Tris resulted in the absence of

proteins in the first four fractions that all proteins were retained by the column in the

starting conditions. The optimized sample preparation led to a separation of the three

chains of the Fibrinogen, particularly the γ-chain of Fibrinogen was eluted much

earlier (F12 and F13) than the α- and the β-chain (F17-F20).

In general, the standard protein mixture was much better separated by applying the

optimized protocol, providing promising starting conditions for the separation of the

protein samples from in vivo biotinylation.

113

PePe

TrypsiTrypsi

LyLy

mar

IN F1 F2 F3 F4 F 5 F6 F7 F 8 F9 F10

F11

mar

IN F12

F13

F14

F 15

F16

F 17

F18

F 19

F20

F 21

F22

T ry p

ma r

2501501007550

37

2520

1510

Fibrinogen

n

psin

sozyme

mar

IN F1 F2 F3 F4 F 5 F6 F7 F 8 F9 F10

F11

mar

IN F12

F13

F14

F 15

F16

F 17

F18

F 19

F20

F 21

F22

T ry p

ma r

2501501007550

37

2520

1510

Fibrinogen

n

psin

sozyme

kDa

Fig. 3.10 SDS-PAGE of the standard protein mixture fractionated by hydroxyapatite chromatography with the optimized protocol

F1 to F12: Fraction 1 to fraction 2 IN: Input: standard protein mixture unfractionated Mar: Marker Tryp: Trypsin

3.3.2 Fractionation kidney protein extract from an in vivo biotinylated mouse

A kidney protein extract from an in vivo biotinylated mouse was fractionated by

hydroxyapatite chromatography applying the optimized protocol (see 3.3.1). The aim

of this experiment was to evaluate wether reduction of the sample complexity by pre-

fractionation with hydroxyapatite chromatography could be achieved.

The protein extract was diluted in sample preparation solution and two equivalent 1

ml samples containing 500 µg protein were prepared and processed as follows:

One sample was submitted to dialysis previous to the loading on the cartridge to

eliminate the phosphate from the lysis buffer in order to check a possible interference

with the hydroxyapatite chromatography. The other was loaded on the cartridge

without previous dialysis.

114

In vivobiotinylatedkidney sample

HA chromatography

SDS-PAGE and SA blot of thefractions

Buffer A ( , 1%SDS, 0.5mM DTE)

Buffer B (500 mM Phosphate, 1%SDS, 7.5 nM CaCl2, 0.5mM DTE)

Conditions:

Dialysis of the sample

5‘ Heating in BufferA(with 100 mM DTE)

Spin dialysis

PO4-

In vivobiotinylatedkidney sample

HA chromatography

SDS-PAGE and SA blot of thefractions

Buffer A ( , 1%SDS, 0.5mM DTE)

Buffer B (500 mM Phosphate, 1%SDS, 7.5 nM CaCl2, 0.5mM DTE)

Conditions:

Dialysis of the sample

5‘ Heating in BufferA(with 100 mM DTE)

Spin dialysis

PO4-

50mM Tris50mM Tris

Fig.3.11 Workflow of the fractionation of the kidney protein

extract from an in vivo biotinylated mouse

Figure 3.12 shows the chromatograms of the undialysed and the dialysed sample.

-500

50100150200250300350400450500

0 3 6 9 12 15 18 21 25 28 31 34 37

ml

mA

U Undialysed SampleDialysed Sample

Fig 3.12 Chromatograms of the HA fractionation of the in vivo biotinylated kidney protein extract (dialysed or not dialysed before loading on the cartridge)

115

The first high peak in the chromatogram of the dialysed sample was much lower than

the one in the chromatogram of the undialysed sample, but it does not contain

proteins. Thus, the elimination of phosphate in the elution buffer did not improve the

separation.

The collected fractions were submitted to a concentration by spin dialysis using

VIVASPIN 500 concentrators (see 2.2.7).

Figure 3.13 shows the SDS-PAGE analysis of the non-dialysed sample fractionated

by hydroxyapatite chromatography.

1515

mar

I N F1 F 2 F3 F 4 F5 F6 F 7 F8 F9 F10

F 11

mar

I N F 12

F 13

F14

F15

F 16

F 17

F 18

F 19

F20

F21/

2 2m

ar

2500

100755037

2520

1510

2500

100755037

2520

1510

kDa

Fig. 3.13 SDS-PAGE of non-dialysed, biotinylated kidney protein extract fractionated by the HA fractionation In: Input: non fractionated, in vivo biotinylated kidney protein extract (20µg

protein) F1 to F21/22: Fraction 1 to fraction 21/22 Mar: Marker

The different band patterns in each lane indicate a successful separation of proteins.

Several intense bands were present in the last three fractions; therefore it seemed

that not all proteins were eluted at the end point of the gradient. The chromatogram

does not reach up to baseline at the end of the run. This is reflected in several

protein bands observed by SDS-PAGE in the last fractions.

Therefore, more optimization steps had to be introduced.

To visualize the separation of biotinylated proteins, the fractions of the protein extract

separated by hydroxyapatite chromatography were submitted to a SA blot analysis

(see Fig. 3.14) applying the following parameters:

116

1. Dialysed, fractionated kidney protein extract:

• Gels: 12% Bis-Tris, 1mm, 15 wells


2. Non-dialysed, fractionated kidney protein extract:

• Gels: 4-12% Bis-Tris, 1mm, 15 wells


ma r

ker

I N F 1 F2 F 3 F4 F 5 F 6 F 7 F 8 F9 F10

F 11

ma r

k er

I N F12

F13

F14

F15

F16

F17

F 18

F 19

F20

F21/

22

ma r

ker

I N F 1 F2 F 3 F4 F 5 F 6 F 7 F 8 F9 F10

F 11

ma r

k er

I N F12

F13

F14

F15

F16

F17

F 18

F 19

F20

F21/

22

UN

DIA

LYSE

D

ma r

ker

I N F 1 F2 F 3 F4 F 5 F 6 F 7 F 8 F9 F10

F 11

ma r

k er

I N F12

F13

F14

F15

F16

F17

F 18

F 19

F20

F21/

22

ma r

ker

I N F 1 F2 F 3 F4 F 5 F 6 F 7 F 8 F9 F10

F 11

ma r

k er

I N F12

F13

F14

F15

F16

F17

F 18

F 19

F20

F21/

22

UN

DIA

LYSE

DD

IALY

SED

kDa kDa

Fig. 3.14 SA blot of the hydroxyapatite chromatography fractions of the dialysed and the non-dialysed kidney protein extract from an in vivo biotinylated mouse In: Input: non fractionated, in vivo biotinylated kidney protein

extract (20µg protein) F1 to F12: Fraction 1 to fraction 21 / 22 Mar: Marker

117

No striking differences were detectable between the SA-blot of the dialysed and the

one of the non-dialysed sample, except of two strong signals in fraction 3 and 4

missing in the SA blot of the undialysed sample.

In both SA blots the biotinylated proteins were limited to the last 15 fractions. The

different signal pattern in each lane indicates a successful separation of the

biotinylated proteins.

In summary, the fractionation of the kidney protein extract from an in vivo biotinylated

mouse was successful. The whole protein extract (see Fig. 3.13) as well as the

biotinylated proteins (see Fig. 3.14) were fractionated by hydroxyapatite

chromatography, as indicated by the different signal patterns in almost each different

lane. However, the introduction of various further optimization steps was necessary

indicated by the presence of a lot of intense signals in the three lanes of the SDS-

PAGE analysis as well as in the SA blot analysis.

3.3.3 Fractionation of a protein extract of a RENCA-bearing kidney from an in vivo biotinylated mouse

Three different optimization steps were tested with the standard protein mixture used

in 3.3.1 (data not shown):

1. A segmented gradient was applied instead of the linear gradient.

2. The phosphate concentration of the buffer B was enhanced to reduce the

protein amount in the last three fractions. (see Fig. 3.13 and Fig. 3.14)

3. The calcium chloride in buffer B was omitted to prevent a blockage of the HA

cartridge by precipitation of calcium chloride.

The optimized protocol described below was applied to a protein extract of a

RENCA-bearing kidney from an in vivo biotinylated mouse.

118

Sample preparation: Heating for 5 minutes in presence of 100

mM DTE and filtration previous to the fractionation

Protein amount: 1.6 mg Buffer A: 50 mM Tris, 1% SDS, 0.5mM DTE (pH7.2) Buffer B: 600 mM Phosphate, 1% SDS, 0.5mM DTE

(pH7.2) Fraction size: 2 ml Flow rate: 0.8 ml / min Linear gradient of 0 – 100 % buffer B within 30 min Segmented gradient part 1: of 0 – 50% buffer B within 7.5min part 2: of 50% -100 % buffer B within 22.5

min

After fractionation of the tumour-kidney protein extract by hydroxyapatite

chromatography, the resulting fractions were concentrated by spin dialysis. Groups of

four fractions were pooled and analysed by SA blot and SDS-PAGE (see Fig. 3.15).

The pooled frations submitted to gel-free proteomic analysis (tryptic on resin

digestion followed by mass spectric analysis).

119

In vivo biotinylated

RENCA sample HA chromatography

SDS-PAGE and SA blot of the fractions

Buffer A (50mM Tris, 1%SDS, 0.5mM DTE)

Buffer B ( , 1%SDS, 7.5 nM CaCl2, 0.5mM DTE)

Conditions:

On resin digestand LC-MS/MS(Giuliano Elia )

Spin dialysis

Pooling

In vivo biotinylatedRENCA sample HA chromatography



Buffer B ( , 1%SDS, 7.5 nM CaCl2, 0.5mM DTE)

Conditions:

On resin digestand LC-MS/MS(Giuliano Elia )

Spin dialysis

Pooling

600mM Phosphate

Linear and segmented gradient

600mM Phosphate

Linear and segmented gradient

Fig.3.15 Workflow of the fractionation of the tumour-kidney protein extract from an in vivo biotinylated mouse

The hydroxyapatite run was performed as specified above in this chapter.

SDS-PAGE of the pooled fractions was performed using the following parameters:

• Gels: 4-12 % Bis-Tris, 1mm, 15 wells


• Protein visualisation: Sypro Ruby stain

Figure 3.16 shows the SDS-PAGE analysis of the RENCA protein extract which was

fractionated by hydroxyapatite chromatographya and whose each four resulting

fractions were pooled together.

120

mar

k er

I N I N (F

+S)

F1-4

F5-8

F9-1

2F1

3-1 6

F1-4

F 5-8

F 9-1

2F 1

3-16

Linear gradient Segmented gradient

250150100755037

2520

1510

mar

k er

I N I N (F

+S)

F1-4

F5-8

F9-1

2F1

3-1 6

F1-4

F 5-8

F 9-1

2F 1

3-16

Linear gradient Segmented gradient

250150100755037

2520

1510

kDa

Fig. 3.16 SDS-PAGE analysis of the tumour-kidney protein extract fractionated by applying a linear or a segmented gradient In: Input: non-fractionated,

untreated protein extract from an in vivo biotinylated mouse (~20µg protein)

In (F+S): non-fractionated protein extract submitted to the sample preparation and to spin dialysis (~20µg protein)

F1-F4 to F13-F16: Pooled fractions 1, 2, 3

and 4 to pooled fractions 13, 14, 15 and 16

Different protein band pattern were detectable in the lanes of the fractions having

applied linear gradient as well as a segmented gradient for the hydroxyapatite

fractionation. This fact indicates a successful separation of the protein extract. The

pooled fractions used a segmented gradient for the fractionation showed more

intense bands pattern, particularly in the lane of the pooled fractions 5 to 8.

Therefore, these pooled fractions were submitted to a shot gun proteomic analysis

performed by Giuliano Elia according to the protocol described in 2.2.11. The shut

gun proteomic analysis did not lead to more protein identifications in the pre-

fractionated protein extract compared to the number of identifications of non-

fractionated protein extract (data not shown).

121

As a consequence of these result, we checked the fractionation of the biotinylated

proteins in this approach by a streptavidin blot using the following parameters:

• Gels: 4 - 12% Bis-Tris, 1mm, 15 wells


Fig. 3.17 shows the streptavidin blot analysis of the pooled and the non-pooled

fractions fractionated by having applied the segmented gradient:

mar

ker

I N F1 F2 F3 F4 F5 F6 F7 F8 F9 F 10

IN( S

+ F)

F11

F 12

F13

F14

F 15

F 16

ma r

ker

I N I N (F

+ S)

F1-4

F5-8

F 9- 1

2F1

3 -16

mar

k er

I N IN( S

+ F)

Pooled FractionsUnpooled Fractions

kDa

Fig. 3.17 SA blot analysis of pooled and non- pooled fractions of the tumour-kidney protein extract fractionated by applying a segmented gradient In: Input: non-fractionated, untreated protein extract from an in vivo

biotinylated mouse (~20µg protein) In (F+S): non-fractionated protein extract submitted to the sample preparation and

to spin dialysis (~20µg protein) F1-F16: Unpooled fractions: from fraction 1 to fraction 16 F1-F4 to F13-F16: Pooled fractions 1, 2, 3 and 4 to pooled fractions 13, 14,

15 and 16

The biotinylated proteins were limited to a few fractions and the most proteins were

present in the lanes of the fractions 5 to 8. These were exactly those fractions which

were pooled for the shut gun proteomic analysis.

122

In summary, the increase of the fraction size and enhancement of the phosphate

concentration in buffer B led to worse performance of the protein fractionation. The

pooling prevented any pre-fractionation previous to the shut gun proteomic analysis.

As consequence, another fractionation of the biotinylated tumour-kidney protein

extract was performed applying the following protocol:

Sample preparation: Heating for 5 minutes in presence of 100

mM DTE and filtration previous to the fractionation

Protein amount: 1.6 mg Buffer A: 50 mM Tris, 1% SDS, 0.5mM DTE (pH7.2) Buffer B: 500 mM Phosphate, 1% SDS, 0.5mM DTE

(pH7.2) Fraction size: 2 ml Flow rate: 0.8 ml / min Linear gradient of 0 – 100 % buffer B within 30 min Segmented gradient part 1: of 0 – 25% buffer B within 3.75 min part 2: of 25 % -100 % buffer B within

26.25 min

After fractionation of the tumour-kidney protein extract, the resulting fractions were

concentrated by spin dialysis, but in difference in this approach the pooling step was

omitted. The resulting fractions were analysed by SA blot (see Fig. 3.18) and were

submitted to a shut gun proteomic analysis (tryptic on resin digestion followed by

mass spectric analysis performed by Giuliano Elia).

123

HA chromatography



Buffer B ( , 1%SDS, 0.5mM DTE)

Conditions: Spin dialysis

Linear and segmented gradientPooling

On resin digestand LC-MS/MS(Giuliano Elia)

In vivo biotinylatedRENCA sample HA chromatography



Buffer B ( , 1%SDS, 0.5mM DTE)

Conditions: Spin dialysis

Linear and segmented gradientPooling

On resin digestand LC-MS/MS(Giuliano Elia)

In vivo biotinylatedRENCA sample

500mM Phosphate500mM Phosphate

Fig. 3.18 Workflow of the fractionation of the tumour-kidney protein extract from an in

vivo biotinylated mouse (optimized protocol)

The degree of pre-fractionation of the tumour-kidney protein extract from an in vivo

biotinylated mouse was checked by SA blot analysis previous to the shut gun

proteomic analysis (see Fig. 3.19). Previous to the blotting the fractions were

submitted to SDS-PAGE with the following parameters:

• Gels: 4 - 12% Bis-Tris, 1mm, 15 wells


Figure 3.19 shows result of this SA-blot analysis:

124

mar

k er

IN F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

F11

mar

ker

IN(S

+F)

F12

F13

F14

F15

F16

F17

F18

F19

F20

I N( S

+F)

F21/

22

IN

LIN

EAR

GR

AD

IEN

TSE

GM

ENTE

D G

RA

DIE

NT

mar

k er

IN F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

F11

mar

ker

IN(S

+F)

F12

F13

F14

F15

F16

F17

F18

F19

F20

I N( S

+F)

F21/

22

IN

LIN

EAR

GR

AD

IEN

TSE

GM

ENTE

D G

RA

DIE

NT

Fig.3.19 SA blot analysis of the tumour-kidney protein extract fractionated by applying a linear or a segmented gradient In: Input: non-fractionated, untreated protein extract from an in

vivo biotinylated mouse (~20µg protein)

In (F+S): non-fractionated protein extract submitted to the sample preparation and to spin dialysis for concentration (~20µg protein)

F1-F16: Unpooled fractions from fraction 1 to fraction 21/22

In these SA blots (see Fig. 3.19) the biotinylated proteins were were present in more

lanes of various fractions compared to the previous SA blot analysis where the

biotinylated proteins were limited to four or five fractions. The biotinylated proteins of

the RENCA protein extract fractionated by applying a linear gradient were present in

more fractions than the biotinylated proteins of the tumour-kidney protein extract

fractionated by applying a segmented gradient. The different signal patterns in the

different lanes of the various fractions indicated a successful fractionation applying

the linear gradient as well as the segmented gradient. The fractions obtained by

fractionation applying the linear gradient were submitted to a shut gun proteomic

analysis (containing an affinity capture on SA sepharose, a tryptic on resin digestion

followed by a mass spectric analysis) performed by Giuliano Elia according to the

protocol 2.2.11.

125

Inpu

t (S+

F)F1

F2F3

F4F5

F6F7

F8F9

F10

F11

F12

F13

F14

F15

F16-

22ID

ENTI

FIED

PR

OTE

INS

IN A

LL F

RA

CTI

ON

SSe

rum

alb

umin

pre

curs

or

Pyru

vate

car

boxy

lase

CBa

sem

ent m

embr

ane-

spec

ific

hepa

ran

sulfa

te p

rote

ogly

can

core

pro

tein

pre

curs

or

Met

hylc

roto

nyl-C

oA c

arbo

xyla

se a

lpha

cha

inO

Sero

trans

ferr

in p

recu

rsor

Gly

cera

ldeh

yde

3-ph

osph

ate

dehy

drog

enas

eL

Type

VI c

olla

gen

alph

a 3

subu

nit

Prop

iony

l-coe

nzym

e A

carb

oxyl

ase,

alp

ha p

olyp

eptid

eU

Lam

inin

alp

ha-5

cha

in p

recu

rsor

Cad

herin

-16

prec

urso

rM

Sodi

um/p

otas

sium

-tran

spor

ting

ATP

ase

beta

-1 c

hain

Serin

e pr

otei

nase

inhi

bito

r A3K

NAl

pha-

1-an

titry

psin

1-4

pre

curs

or

Plas

ma

glut

athi

on p

erox

idas

e pr

ecur

sor

4F2

cells

urfa

ce a

ntig

en h

eavy

cha

inW

Lam

inin

gam

ma-

1 ch

ain

prec

urso

r

Acet

yl-C

oA c

arbo

xyla

se 2

65 (F

ragm

ent)

ATr

anst

hyre

tin p

recu

rsor

Alph

a-2-

mac

rogl

obul

in p

recu

rsor

SC

olla

gen

alph

a 2

(VI)

chai

n pr

ecur

sor

Sim

ilar t

o fib

rinog

en, g

amm

a po

lype

ptid

e

Vita

min

D-b

indi

ng p

rote

in p

recu

rsor

BFi

brin

ogen

, B b

eta

poly

pept

ide

Hea

t sho

ck p

rote

in H

SP

90-a

lpha

RAc

tin, c

ytop

lasm

ic 1

Tubu

lin a

lpha

-1 c

hain

OTu

bulin

bet

a-2

chai

n

Mac

roph

age

mig

ratio

n in

hibi

tory

fact

orK

Apol

ipor

prot

ein

A-I

prec

urso

r

Inte

grin

bet

a-1

prec

urso

rE

Lum

ican

pre

curs

or

Lam

inin

bet

a-1

chai

n pr

ecur

sor

NC

olla

gen

alph

a 1

(XV

III) c

hain

pre

curs

or

Col

lage

n al

pha

2(IV

)

Ecto

nucl

eosi

de tr

ipho

spha

te d

ipho

spho

hydr

ogel

ase

1

L-gl

ycer

in-r

ich

glyc

opro

tein

His

tidin

e-ric

h gl

yco

prot

ein

Tabl. 3.1 Protein identifications from the different fractions and from the non-fractionated tumour-kidney protein extract from an in vivo biotinylated mouse

: indicates the identification of the corresponding protein in this fraction F1-F21/22: fraction 1 to fraction 21/22

126

In the shut gun proteomic analysis, only few more proteins were identified in the

sample obmitted to pre-fractionation compared to the unprocessed sample. In fact,

the proteins identified were different in the two samples. Proteins were identified

mostly in one single fraction indicating a successful fractionation of such a complex

protein mixture by hydroxyapatite chromatography. This fact confirms the conclusion

drawn from the SA blot.

Exceptions were trypsin and albumin nearly present in all fractions. It should be

noted that this two proteins were artifacts from either the perfusion procedure

(albumin, the most abundant blood protein) or from the tryptic on resin digestion.

127

3.4 In vivo biotinylation of tumour-bearing mice with sulfo-NHS-SS-biotin as alternative biotinylation reagent

In the gel-free proteomic analysis of biotinylated proteins from protein extracts from in

vivo biotinylated mice, in addition to the expected proteins from cell plasma

membrane and extracellular matrix, several of intracellular proteins were identified

(see 3.3.3 and not shown data from Giuliano Elia). Even though the biotinylation

reagent carried a charged sulfate group, obviously it could penetrate biological

membranes to a certain extent. To overcome this problem instead of sulfo-NHS-LC-

biotin the cleavable, disulfide containing Sulfo-NHS-SS-biotin was used as

biotinylation reagent. Peirce and co-workers (Peirce et al, 2004) observed that cell

surface biotinylation of permeabilized cells with Sulfo-NHS-SS-biotin lead in a

significant proportion to biotinylation of the whole intracellular compartment, while this

was not the case when Sulfo-NHS-SS-biotin was used as biotinylation reagent. This

may be a result of cleavage of the disulfide bond due to the reducing intracellular

environment.

To reduce the contamination with intracellular proteins, we set out the perfusion with

sulfo-NHS-SS-biotin instead of the sulfo-NHS-LC-biotin. To apply the same molar

concentration of biotinylation reagent (~1.8 mM) 1.09 mg/ml of Sulfo-NHS-SS-biotin

was used.

In a pre experiment, in which Black six mice had been perfused with Sulfo-NHS-SS-

biotin using the perfusion conditions mentioned below, there was detected by

histochemical analysis that an in vivo biotinylation with this alternative biotinylation

reagent is feasible (data not shown).

After this pre experiment F9 tumour-bearing SvEv/129 mice were submitted to in vivo

biotinylation according to the protocol described in 2.2.2 using the following perfusion

conditions:

128

Perfusion needle

Perfusion pressure

Perfusion flow rate


solutions

Warming the

animal

1. Perfusion

step: washing

2. Perfusion

step: biotinylation

3. Perfusion

step: quenching

Perfusion needle

In vivo biotinylation with Sulfo-NHS-SS-

Biotin

25G without

small tube

100 mm Hg

~1.8 ml / min 38°C Heating pad and IR-lamp

No 15 ml Sulfo-NHS-SS-Biotin

1.09 mg/ml,

10% (w/v) Dextran 40

in PBS pH 7.4

10 min 50 mM

Tris, 10% (w/v)

Dextran 40

in PBS

After the perfusion the organs of interest (liver, kidney, muscle and tumour) were

excised, embedded in cryomedium and snap-frozen. Section from liver, kidney

muscle and tumour were cut and submitted to histochemical analysis with

Streptavidin:biotinylated alkaline phosphatase/FAST-Red TR (red staining of

biotinylated structures) and counterstained with Haematoxilin (blue staining)

according to the protocol described in 2.2.4 (data only shown for tumour).

Figure 3.20 shows tumour sections representative for the above, described perfusion

experiment. In addition for comparison a tumour section, which derived from a mouse

perfused with sulfo-NHS-LC-biotin according to the optimized perfusion protocol:

Perfusion

needle Perfusion pressure

Perfusion flow rate


solutions

Warming the animal

1. Perfusion

step: washing

2. Perfusion

step: biotinylation

3. Perfusion

step: quenching

Perfusion needle

Final optimized protocol

25G without

small tube

100 mm Hg

~1.8 ml / min 38°C Heating pad and IR-lamp

No 15 ml Sulfo-NHS-LC-

Biotin 1 mg/ml,

10% (w/v) Dextran 40

in PBS pH 7.4in

PBS

10 min 50 mM

Tris, 10% (w/v) Dextran

40 in PBS

In general, the tumour sections, derived from organs which were successfully

biotinylated with Sulfo-NHS-SS-biotin, indicated a weaker red staining than the

tumour sections derived from mice perfused with sulfo-NHS-LC-biotin. This indicates

that accessible vascular structures were biotinylated to a lesser extent compared with

sulfo-NHS-LC-biotin. This fact could be explained with the minor stability of the sulfo-

NHS-SS-biotin as consequence of its disulfide bound.

129

A B C

D D

A B C

D D

Fig. 3.20 Histochemical detection of biotinylated structures of tumour sections from mice perfused with sulfo-NHS-SS-biotin or sulfo-NHS-LC-biotin A tumour section from mouse perfused with Sulfo-NHS-

SS-biotin): partly stained B tumour section from a mouse perfused with Sulfo-

NHS-SS-biotin: partly stained C tumour section from a mouse pefused with Sulfo-

NHS-SS-biotin: not stained at all D tumour sections from a mouse perfused with Sulfo-

NHS-LC-biotin

The specimens of all of organs of interest (kidney, liver, muscle and tumour) from

mice perfused with Sulfo-NHS-SS-biotin, whose tumour sections had shown a

positive result in the histochemical analysis, were homogenised in 20 µl lysis buffer

per mg tissue according to the protocol described in 2.2.5.1. Total protein extracts

were prepared as described in 2.2.5.2 and the protein concentration was determined.

The protein extracts were diluted in lysis buffer and, for each of them 24 µl

(corresponding to 100 µg protein) were subjected to a SA blot analysis.

Figure 3.21 shows the SA blot analysis of biotinylated proteins, recovered from

homogenates of muscle (M), liver (L), kidney (K) and tumour (T), from mice, which

had been perfused with sulfo-NHS-SS-biotin of sulfo-NHS-LC-biotin (C+).

In the SA blot, which can only detect biotinylated proteins, signals were present in all

lanes indicating a successful biotinylation. The fact that the signal pattern of the “C+”

130

lane was more intense than the signal pattern present in all the other lanes indicates

that the perfusion with sulfo-NHS-LC-biotin led to more biotinylated proteins than the

perfusion with sulfo-NHS-SS-biotin. This may be explained with the lower stability of

the sulfo-NHS-SS-biotin compared to the sulfo-NHS-LC-biotin.

mar

ker

C+

T 1 M1

L 1

K1

T2 M2

L2 K2

160

10575

50

3530

1525

10

mar

ker

C+

T 1 M1

L 1

K1

T2 M2

L2 K2

160

10575

50

3530

1525

10

kDa

Fig. 3.21 SA blot analysis of biotinylated proteins, recovered from homogenates of muscle (M), liver (L), kidney (K) and tumour (T), from mice, which had been perfused with sulfo-NHS-SS-biotin of sulfo-NHS-LC-biotin (C+).

The protein extracts of all organs of interest (kidney, liver, muscle and tumour) from

mice perfused with Sulfo-NHS-SS-biotin, whose tumour sections had shown a

positive result in the histochemical analysis were submitted to a shot gun proteomic

anaylsis. This shot gun proteomic analysis, performed by Giuliano Elia as described

in 2.2.11, featured an affinity capture on SA sepharose, a tryptic on resin digestion

and mass spectrometric analysis of the resulting peptides (data not shown). The fact

that fewer proteins were identified compared to previous shot gun proteomic analysis

of protein extracts from mice perfused with sulfo-NHS-LC-biotin confirms the

conclusion drawn from the SA blot analysis. Fewer proteins were biotinylated with the

sulfo-NHS-SS-biotin than with sulfo-NHS-LC-biotin, which could be explained with its

lower stability of the biotinylation reagent.

131

132

A reduction of the ratio of cytosolic protein was not detectable compared to the

previous protein identifications obtained from protein extracts derived from mice

perfused with Sulfo-NHS-LC-biotin.

3.5 Synthesis of a dextran-biotin-NHS derivative as an alternative less cell penetrating perfusion reagent

In the gel-free proteomic analysis of biotinylated proteins from protein extracts from in

vivo biotinylated mice, in addition to the expected proteins from cell plasma

membrane and extracellular matrix, several intracellular proteins were identified (see

3.3 and not shown data from Giuliano Elia). Even though the biotinylation reagent

carried a charged sulfate group, obviously it could penetrate biological membranes to

a certain extent. To overcome this problem, a dextran-biotin-NHS derivative was

synthetized, providing the following features: 1) biotin residues for affinity capturing

on streptavidin, 2) NHS groups for the reaction with primary amines, 3) a fluorescent

label for the direct detection by fluorecent microscopy of the corresponding organ

sections or the direct detection of protein gel bands in a fluorescence imager, and 4)

a high molecular weightcompound, it should not be able to penetrate biological

membranes.

Therefore, a commercially available 70 kDa dextran modified with 5 lysins and 40

biotins was reacted with a 100 x excess of disuccinimidyl tartrate (DST), which acts

as crosslinker of primary amino groups (see Fig. 2.17). The excess of crosslinker was

used in order to reach the reaction of each dextran lysine residue with another DST

molecule, avoiding crosslinking of amino groups in the same or in different dextran

molecules.

3.5.1 Pilot experiment: Elimination of the crosslinker simulated by Fluorescein

After the synthesis reaction described above, the excess DST had to be eliminated.

In a pilot experiment the purification of high molecular weight dextran from a low

molecular weight compound like DST was tested. To facilitate the analysis of the

purification, yellow coloured Fluorescein was used in this experiment to mimic the

DST which was planned to use in the real synthesis.

133

Dextran was labelled with fluorescein-5-isothiocyanate as described in 2.2.12.2.1.

Four different samples were prepared:

1) Labelled Dextran 250 µl 40 µM dextran in MilliQ, 12.5 µl Fluorescein-5-

isothiocyanate and 237.5 µl DMSO were incubated for 2 h at room temperature

(labelling reaction).

2) Labelled Dextran with an excess of Fluorescein: 25 µl 40 µM dextran in MilliQ,

50 µl 2 mM Fluorescein-5-isothiocyanate in DMSO, 122,5 µl 40 mM Fluorescein in

DMSO, 77.5 µl DMSO and 225 µl MilliQ were incubated for 2 h at room temperature

(labelling reaction).

3) Unlabelled Dextran with an excess of Fluorescein: 25 µl 40 µM dextran in

MilliQ, 125 µl 40 mM Fluorescein in DMSO, 125 µl DMSO and 225 µl MilliQ

4) Fluorescein: 125 µl 40 mM Fluorescein in DMSO, 250 µl MilliQ and 125 µl DMSO

These four samples were submitted to four different purification attempts (Fig. 3.22):

I) spin dialysis according to 2.2.12.3.1 with 50% (v/v) DMSO in water as diluent

II) spin dialysis according to 2.2.12.3.1 with PBS as diluent

III) gel filtration (PD-10) according to 2.2.12.3.2 using PBS as equilibration and

elution solution

IV) gel filtration (PD-10) according to 2.2.12.3.2 using 50% DMSO in water as

equilibration and elution solution

134

•50%DMSO/50%Water

•50%DMSO/50%Water

•PBS

•PBS

F

1. B

Labeled dextran

2.

F

B

Labeled dextranwith an excess of fluorescein

3.B

Unlabeled dextranwith an excess of fluorescein Fluorescein

4.

Spindialysis

PD-10

•50%DMSO/50%Water

•50%DMSO/50%Water

•PBS

•PBS

F

1. B

Labeled dextran

2.

F

B

Labeled dextranwith an excess of fluorescein

3.B

Unlabeled dextranwith an excess of fluorescein Fluorescein

4.

Spindialysis

PD-10

Fig.3.22 Scheme of the different approaches for the elimination of

Fluorescein from the modified dextran by PD-10 and spin dialysis (See text for explanation.)

Figure 3.23 shows the results of purification attempt I, in which the four approaches

were each submitted to spin dialysis with VIVASPIN 500 concentrators according to

the protocol described in 2.2.12.3.1. After applying the sample to the sample

reservoir, the first centrifugation step was performed for 5 min at 14’000 × g and the

concentrate was diluted to 500 µl. Centrifugation and dilution were repeated four

more times. Each flow through sample and the final concentrate sample were

collected and the absorption at 490 nm was measured with a plate reader. The

results were plotted in a bar chart.

In the spin dialysis of labelled dextran alone (Fig. 3.23. A), there was no absorbance

detected in the flow through after the first three centrifugation steps. This was

expected as the fluorescently labelled dextran should not pass through the dialysis

membrane (MW cut off = 10 kDa) due to its high molecular weight (70 kDa).

However, after the third centrifugation step the flow through contained fluorescence,

indicating the disintegration of the dialysis membrane, maybe due to the DMSO

content in the dilution solution. When labelled dextran in combination with a 100x

excess of non-reactive fluorescein was submitted to the same purification procedure,

there was absorption detected in the first four flow through, deriving from low

molecular weight fluorescein. However, even after the forth dilution and centrifugation

step, the final concentrate still exhibited such a high absorbance leading to the

conclusion that even after three runs of spin dialysis the dextran cannot be freed from

135

the excessive low molecular weight compound in the chosen conditions. This

conclusion is supported also by the results of approach 3 (unlabeled dextran +

excess of fluorscein) which showed high absorption not only in flow through 1-3 but

also in the final concentrate. (The absence of absorption in flow through 4 is strange

and may be explained by clogging of the filter membrane by the dextran). In contrast

to these findings, in the spin dialysis of fluorescein alone fluorescence was detected

only in the flow throughs but not in the final concentrate. This apparent removal of the

low molecular weight compound seemed to be hindered when dextran is present in

the sample. In conclusion, purification of dextran from a low molecular weight

compound using spin dialysis and dilution with 50% DMSO in water failed.

A0.000

0.100

0.200

0.300

0.400

0.500

FT 1 FT 2 FT 3 FT 4 ED

Abs

B2.3002.3502.4002.4502.5002.5502.600


Abs

C 0.0000.5001.0001.5002.0002.5003.000


Abs

D 0.0000.5001.0001.5002.0002.5003.000


Abs

A0.000

0.100

0.200

0.300

0.400

0.500


Abs

B2.3002.3502.4002.4502.5002.5502.600


Abs

C 0.0000.5001.0001.5002.0002.5003.000


Abs

D 0.0000.5001.0001.5002.0002.5003.000


Abs

Fig.3.23 Attempt I for the purification of dextran from a low molecular weight compound: spin dialysis according to 2.14.2.1 with 50% (v/v) DMSO in water as diluent A: Labelled Dextran B: Labelled Dextran + excess of Fluorescein C: Unlabelled Dextran + excess of Fluorescein D: Fluorescein FT 1: Flow through of the first centrifugation step FT 2: Flow through of the second centrifugation step FT 3: Flow through of the third centrifugation step FT 4: Flow through of the fourth centrifugation step ED: final diluted concentrate (See text for explanation.)

136

Figure 3.24 shows the results of purification approach II, in which the four samples

were each submitted again to the spin dialysis procedure described above. In

contrast to purification attempt I PBS was used as diluent instead of 50 % (v/v)

DMSO in MilliQ.

In the purification approach of the labelled dextran alone (Fig 3.x A), there was little

absorbance found in all four flow throughs. The highest absorption was detected in

the final dilution. The absorbance in the flow throughs is in conflict with the fact the

fluorescently labelled dextran should absolutely not pass through the dialysis

membrane (MW cut off = 10 kDa) as a consequence of its high molecular weight (70

kDa). Therefore, the absorbance detected in the flow throughs indicated the

disintegration of the dialysis membrane, maybe as a result of the 50 % (v/v) DMSO in

MilliQ of the original sample.

When labelled dextran mixed with a 100x excess of non-reactive fluorescein was

submitted to the same spin dialysis procedure, there was absorption detected in the

first four flow throughs, which was so high, that it had to derive mostly from the low

molecular weight Fluorescein. The final dilution still showed such a high absorbance

indicating that the dextran cannot be freed from all of the excessive low molecular

Fluorescein by applying this spin dialysis procedure. This conclusion is confirmed by

the results of approach 3 (unlabeled dextran + excess of fluorscein) which showed

high absorption not only in flow through 1-4, but also in the final diluted concentrate.

In the spin dialysis of Fluorescein alone, fluorescence was detected only in the four

flow throughs, but not in the final concentrate. The elimination of the Fluorescein

seemed to be prevented when dextran was added to the sample, even though PBS

was used as diluent.

In summary, purification of dextran from a low molecular weight compound using spin

dialysis did not work in our hands, regardless of which solution was used as diluent.

137

A0.000

0.100

0.200

0.300

0.400

0.500


Abs

0.0000.5001.0001.5002.0002.5003.000


Abs

B

C0.0000.5001.0001.5002.0002.5003.000


Abs

D0.0000.5001.0001.5002.0002.5003.000


Abs

Fig.3.24 Attempt II for the purification of dextran from a low molecular weight compound: spin dialysis according to 2.14.2.1 with PBS as diluent A: Labelled Dextran B: Labelled Dextran + excess of Fluorescein C: Unlabelled Dextran + excess of Fluorescein D: Fluorescein FT 1: Flow through of the first centrifugation step FT 2: Flow through of the second centrifugation step FT 3: Flow through of the third centrifugation step FT 4: Flow through of the fourth centrifugation step ED: final diluted concentrate (See text for explanation.)

Figure 3.25 shows the results of purification attempt III, in which the four samples

were each submitted to gel filtration with pre-packed PD-10 desalting columns

according to the protocol described in 2.2.12.3.2. After equilibration of the PD-10

column the sample was applied and completed to 2,5 ml with elution buffer. The four

samples were eluted from the column with up to 30 ml elution solution (50 % DMSO

in water), 0.5 ml fractions were collected and the absorption was measured with a

plate reader. The results were plotted in a line chart.

In the gel filtration of labelled dextran alone (Fig 3.25 A), there was only one peak

present in the range of the fractions 7 to 14 indicating a successful labelling reaction.

138

An elution of the fluorescently labelled dextran in this range of the fractions was

expected due to its high molecular weight (70 kDa).

When labelled dextran in combination with a 100x excess of non-reactive Fluorescein

was submitted to this gel filtration procedure, there was only one huge peak present

in the range of fraction 6 to 26 compared to the low molecular weight component

Fluorescein. The small peak derived from the fluorescently labelled dextran clearly

detectable in Fig. 3.25 A was totally hidden behind the huge peak of the fluorescein

in Fig 3.25.B.

This conclusion is supported also by the results of approach 3 (unlabeled dextran +

excess of fluorscein) and 4 (fluorescein alone), as the chromatograms of all three

approaches (2, 3 and 4) looked similar showing only the huge peak of the

Fluorescein independent of the additional presence of labeled dextran.

In conclusion, purification of dextran from a low molecular weight compound using

gel filtration with 50% DMSO in water as equilibration and elution solution failed.

139

-0.050

0.000

0.050

0.100

0.150

0 5 10 15 20 25 30 35

Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.5003.000

0 5 10 15 20 25 30 35Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.500

0 5 10 15 20 25 30 35

Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.5003.000

0 5 10 15 20 25 30Fractions

Abs

A B

C D

-0.050

0.000

0.050

0.100

0.150

0 5 10 15 20 25 30 35

Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.5003.000

0 5 10 15 20 25 30 35Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.500

0 5 10 15 20 25 30 35

Fractions

Abs

-0.5000.0000.5001.0001.5002.0002.5003.000

0 5 10 15 20 25 30Fractions

Abs

A B

C D

Fig.3.25 Attempt III for the purification of dextran from a low molecular weight compound: gel filtration by PD-10 desalting columns according to 2.14.2.2 with 50 % (v/v) DMSO in water as elution and equilibration solution A: Labelled Dextran B: Labelled Dextran + excess of Fluorescein C: Unlabelled Dextran + excess of Fluorescein D: Fluorescein (See text for explanation.)

Figure 3.26 shows the results of purification attempt IV, in which the four approaches

were each again submitted to gel filtration according to the protocol described in

2.2.12.3.2. In contrast to purification attempt III where 50 % (v/v) DMSO in water was

used as equilibration and elution solution, PBS was applied as equilibration and

elution solution.

In the gel filtration of labelled dextran alone (Fig 3.26 A), there was only one peak

present like in Fig 3.26 A in the range of fraction 7 to 14. Due to the high molecular

weight of the fluorescently labelled dextran its elution at this area of the

chromatogram was expected. The absence of a peak derived from the free labelling

reagent (Fluorescein-5-isothiocyanate) (which should be eluted later) indicated the

successful labelling reaction.

When labelled dextran in combination with a 100x excess of non-reactive Fluorescein

was submitted to gel filtration, there were two different peaks present in the

140

chromatogram: one small peak in the range of fraction 7 to 12 (highlighted with a red

arrow) which derived from the early diluted fluorescently labelled dextran and a huge

one which begins to increase from fraction 14 on, indicating the presence of an

excess of the low molecular weight component Fluorescein.

The conclusion that the small peak really derived from the fluorescently labelled

dextran was confirmed by the results of approach 3 (unlabeled dextran + excess of

fluorscein) and 4 (fluorescein alone), as the chromatograms of this approaches (C

and D ) looked similar, while showing only the huge peak of the Fluorescein

independent of the additional presence of the unlabelled dextran. The similarity of

those two chromatograms indicates that the dextran did not interact with the material

of the PD-10 column and therefore, did not hinder a successful separation of the low

molecular weight compound Fluorescein from the high molecular weight compound

dextran.

-0.050

0.000

0.050

0.100

0.150

0 10 20 30 40 50 60

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0 5 10 15 20

Fraction

Abs

-0.050

0.0000.050

0.100

0.1500.200

0.250

0 5 10 15 20

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0 5 10 15

Fractions

Abs

Fractions Fractions

FractionsFractions

Abs

Abs

Abs

Abs

-0.050

0.000

0.050

0.100

0.150

0 10 20 30 40 50 60

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0 5 10 15 20

Fraction

Abs

-0.050

0.0000.050

0.100

0.1500.200

0.250

0 5 10 15 20

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0 5 10 15

Fractions

Abs

Fractions Fractions

FractionsFractions

Abs

Abs

Abs

Abs

Fig.3.26 Attempt IV for the purification of dextran from a low molecular weight compound: gel filtration by PD-10 desalting columns according to 2.14.2.2 with 50 % (v/v) DMSO in water as elution and equilibration solution A: Labelled Dextran B: Labelled Dextran + excess of Fluorescein C: Unlabelled Dextran + excess of Fluorescein D: Fluorescein (See text for explanation.)

141

In conclusion, a purification of dextran from a low molecular weight compound (e.g.

fluorescein) could be achieved using gel filtration with PBS as equilibration and

elution buffer succeded.

3.5.2 Synthesis, purification and activity test of the dextran-biotin-NHS derivative

After the synthesis reaction and elimination of the excessive DST by PD-10 the NHS-

groups of the dextran-biotin-NHS derivative have still to react with primary amines.

Therefore, the reactivity of the purified dextran-biotin-NHS derivative was tested by

addition of Fluoresceinamine, which was expected to react with the NHS-groups of

the dextran-derivative. Additionaly, also the possible presence of free DST

(disuccinimidyl tartrate) should be detecteble by applying this activity test, as the free

crosslinker also react with the mentioned Fluoresceinamine.

Dextran was labelled with fluorescein-5-isothiocyanate as described in 2.2.12.2.1.

The synthesis reaction of the labelled dextran with a 100 fold excess of DST was

perfomed as follow:

625 µl 40 mM DST in DMSO and 250 µl 25 µM fluorescently labelled dextran in 50 %

(v/v) DMSO in water were incubated for 15min at room temperature. This synthesis

reaction was repeated another two times and the resulting samples were submitted

to different experiments:

1) The first synthesis mixture was submitted to a gel filtration with PB as elution

and equilibration solution according to the protocol described above and the

absorption was measured with a plate reader to identify the fractions

containing the labelled dextran-biotin-NHS derivative. The results were plotted

in a line chart. (see Fig. 3.27, picture 3.)

2) After addition of 30 µl 20 mM Fluoresceinamine in 50 % (v/v) DMSO in PB to

the second synthesis mixture a gel filtration with PB as elution and

equilibration solution was performed according to the protocol described

above. The result of the absorption measurement were plotted in a line chart.

(see Fig.3.27, picture 4.)

142

3) The synthesis mixture was submitted to a gel filtration with PB as elution and

equilibration solution according to the above described protocol. To each of

the fractions 8 to 12 (which had been identified as fractions containing the

labelled dextran-biotin-NHS derivative see 1) above) 5 µl 20 mM

Fluoresceinamine in 50 % DMSO in PB was added. These fractions were

pooled and another gel filtration was performed according to the protocol

described above. The results of the absorption measurement were plotted in a

line chart. (see Fig.3.27, picture 5.)

Fluoresceinamin Fluoresceinamin+ DST

Labeled dextran with DST+ DST

1.F

NH2

F NH2

1.FF

NH2

F NH2F NH2F NH2

2.2. 3.3.

PD-10 PD-10 PD-10

-0.050

0.000

0.050

0.100

0.150

0.200

0 5 10 15 20 25 30 35 40

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

-0.020

0.000

0.020

0.040

0.060

0 5 10 15 20 25 30

Fraction

Abs

ABSORPTION MEASUREMENT

Fluoresceinamin Fluoresceinamin+ DST

Labeled dextran with DST+ DST

1.F

NH2

F NH2

1.FF

NH2

F NH2F NH2F NH2

2.2. 3.3.

PD-10 PD-10 PD-10

-0.050

0.000

0.050

0.100

0.150

0.200

0 5 10 15 20 25 30 35 40

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

-0.020

0.000

0.020

0.040

0.060

0 5 10 15 20 25 30

Fraction

Abs

ABSORPTION MEASUREMENT

Dextran+Crosslinker

PD-10

ABSORPTION MEASUREMENT -0.0200.0000.0200.0400.0600.0800.1000.1200.1400.160

0 5 10 15 20 25 30

Fractions

Abs

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

F NH2F NH2F NH2

F NH2F NH2F NH2

4. 5.

Dextran+Crosslinker

PD-10

ABSORPTION MEASUREMENT -0.0200.0000.0200.0400.0600.0800.1000.1200.1400.160

0 5 10 15 20 25 30

Fractions

Abs

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

+

PD-10

Fluoresceinamin

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

F NH2F NH2F NH2

F NH2F NH2F NH2

4. 5.

Fig.3.27 Overview of the different approaches with their further processing See text for explanation.

143

Two further approaches were prepared as follows:

4) 10 µl 20 mM Fluoresceinamine in 50 % (v/v) DMSO in PB and 490 µl PB were

incubated for 15 min and submitted to a gel filtration according to the protocol

described above. The results of the absorption measurement were plotted in a

line chart. (see Fig.3.27 picture 1.)

5) 250 µl 40 mM DST in DMSO and 10 µl 20 mM Fluoresceinamine in 50 %

DMSO in PB were incubated for 15 min at room temperature and submitted to

a gel filtration according to the protocol described above. The results of the

absorption measurement were plotted in a line chart. (see Fig.3.27 picture 2.)

Figure 3.28 shows the results of the activity test.

The chromatogram of the Fluoresceinamine exhibits only one huge peak increasing

from fraction 25 on.

When DST in combination with Fluoresceinamine was submitted to a gel filtration,

there were two various peaks detectable: one smaller peak from DST reacted

Fluoresceinamine and a huge peak derived from the later eluted free

Fluoresceinamine.

The conclusion that the first smaller peak stemmed from the Fluoresceinamine

reacted with DST was confirmed by direct comparison of this chromatogram with the

chromatogram (Fluoresceinamine alone) described above where this smaller peak

was missing.

In the gel filtration of labelled dextran-NHS derivative with an excess of DST (Fig

3.28, C), there was only one peak present in the range of fraction 7 to 14. Due to the

high molecular weight of the fluorescently labelled dextran-biotin-NHS derivative its

elution at this area of the chromatogram was expected. The absence of a peak

derived from the excessive DST reagent was expected as a consequence of its

excitation by a lower wavelength than 490 nm.

When the fluorescently labelled dextran-NHS derivative without previous purification

was mixed with Fluoresceinamine, there were three different peaks detectable:

The first eluting and smallest peak derived from the labelled dextran-NHS derivative

(highlighted with a red arrow), the second peak stemmed from the Fluoresceinamine

144

reacted with DST and the last huge peak showed the presence of the free

Fluoresceinamine in the sample.

-0.050

0.000

0.050

0.100

0.150

0.200

0 5 10 15 20 25 30 35 40

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

-0.020

0.000

0.020

0.040

0.060

0 5 10 15 20 25 30

Fraction

Abs

-0.500

0.000

0.500

1.000

1.500

0 5 10 15 20 25 30

Frations

Abs

-0.0200.0000.0200.0400.0600.0800.1000.1200.1400.160

0 5 10 15 20 25 30

Fractions

Abs

A

B

C

D

E

-0.050

0.000

0.050

0.100

0.150

0.200

0 5 10 15 20 25 30 35 40

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

-0.020

0.000

0.020

0.040

0.060

0 5 10 15 20 25 30

Fraction

Abs

-0.500

0.000

0.500

1.000

1.500

0 5 10 15 20 25 30

Frations

Abs

-0.0200.0000.0200.0400.0600.0800.1000.1200.1400.160

0 5 10 15 20 25 30

Fractions

Abs

A

B

C

D

E

-0.050

0.000

0.050

0.100

0.150

0.200

0 5 10 15 20 25 30 35 40

Fractions

Abs

-0.050

0.000

0.050

0.100

0.150

0.200

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fractions

Abs

-0.020

0.000

0.020

0.040

0.060

0 5 10 15 20 25 30

Fraction

Abs

-0.500

0.000

0.500

1.000

1.500

0 5 10 15 20 25 30

Frations

Abs

-0.0200.0000.0200.0400.0600.0800.1000.1200.1400.160

0 5 10 15 20 25 30

Fractions

Abs

A

B

C

D

E

Fig. 3.28 Activity test of the Dextran-biotin-NHS derivative with Fluoresceinamine A: Fluoresceinamine B: Fluoresceinamine + excess of DST C: Labelled dextran-NHS derivative + excess of DST D: Labelled dextran-NHS derivative + excess of DST + Fluoresceinamine E: Labelled dextran-NHS-derivative purified by PD-10 and Fluoresceinamine

In the chromatogram of approach 5 (purified dextran-biotin-NHS derivative +

Fluoresceinamine), there were two different peaks detectable: a small peak derived

from the labelled NHS-derivative (highlighted by red arrows) and another huge peak

indicating the presence of free Fluoresceinamine. The absence of the peak derived

from Fluoresceinamine indicates the successful elimination of DST by gel filtration

with PB as elution and equilibration solution. The low height of the first peak indicates

the moderate reactivity of the NHS-goups of the dextran-derivative against the amine

– group of the Fluoresceinamine as no significant enhancement of the absorption of

145

146

this peak was detectable, compared to the peak of the labelled dextran derivative in

chromatogram C (labelled dextran-biotin-NHS derivative).

From the result of figure 3.28, we can conclude that only a low fraction of primary

amines (e.g., Fluoresceinamin) can be incorporated into dextran. In spite of a good

elimination of DST from the dextran solution through PD-10 chromatography, in

essence the procedure was judged to be not suitable for the high demands (e.g.,

quality, cost) of in vivo perfusion procedure.

4 DISCUSSION The aim of the diploma thesis was the optimisation of the in vivo biotinylation

technology which was recently set up in our laboratory.

This method aims at the identification of novel tissue-specific markers readily

accessible from the blood, which could serve as targets for selective drug

delivery and for diagnostic imaging of certain disorders (see 1.1).

Several attempts have been made for the discovery of vascular targets in

tumours comprising different in vitro and in vivo models [overview in Scheurer

et al, 2004]. In vitro models are used for the identification of significant

changes in the gene or protein expression in response to environmental

changes. These models mimic the tumour environment which is characterized

by the presence of new blood vessels and the insufficient blood perfusion of

the tumour mass, leading to hypoxia, serum starvation and pH changes. The

advantage of this kind of models is their reduced complexity with respect to

the in vivo situation, which allows appreciating the effect of modifying a single

variable at a time. However, results based on these experiments are difficult

to transfer to real tumours in an organism, because the situation in the tumour

is influenced by a complex interplay of parameters.

In principle, the most direct way to assess differences in the accessibility of

proteins in different types of tissue consists in the in vivo labeling of vascular

structures, followed by rapid recovery and comparative proteomic analysis of

the proteins.

This strategy was, e.g., chosen by the group of Schnitzer [Jacobson et al.,

1992; Czarny et al., 2003] using the in vivo coating of vascular structures with

colloidal silica in tumours and in normal organs and recovery (by

centrifugation and fractionation) of silica-coated structures (luminal cell

plasma membranes and caveolae of the endothelium). Any approach,

Schnitzer and coworkers recently reported on the “Subtractive proteomic

147

mapping of the endothelial surface in lung and solid tumours for tissue-

specific therapy“[Oh et al., 2004] and the identification of a number of

accessible tissue-markers. Aminopeptidase-P and annexin A1were found as

selective in vivo targets for antibodies in lungs and solid tumours,

respectively. However, their choice of 2D-PAGE for the proteomic analysis

seems questionable as, at least in our experience, hydrophobic membrane

proteins may be excluded. Another in vivo approach was pioneered by

Ruoslahti, Pasqualini and co-workers: The in vivo biopanning of peptide

phage libraries aimes at the identification of peptides which specifically bind

different vascular addresses in different tissues and/or tumours [Rajotte et al.,

1998; Pasqualini and Rouslahti, 1996]. The potential of this technology,

remains to be demonstrated, considering the absence of quantitative

biodistribution studies and clinical studies with purified preparations of the

selected vascular-targeting peptides.

Inspired by the work of De La Fuente and co-workers [De La Fuente et al.,

1997], who performed ex vivo perfusion of isolated rat lungs with a

biotinylation reagent (see 1.2.2), Rybak et al. [manuscript in preparation] have

set up in our laboratory a novel in vivo technology – termed “in vivo

biotinylation” – for the discovery of tissue-specific targets accessible from

circulation (see 1.3). However, this ongoing project posed several problems,

some of which were tackled in the frame of this diploma thesis.

4.1 Perfusion of tumours Research on marker discovery and selective drug delivery is of special

importance for the development of better tumour diagnosis and therapy

strategies (see 1.1.1).

Thus, in vivo biotinylation was applied to mice bearing a subcutaneous tumour

in order to seek for proteins which are more abundantly expressed or better

accessible in tumours than in healthy tissues.

148

However, the perfusion of tumours was much less efficient and more

heterogeneous compared to other organs (see 3.1). Overall, with the original

perfusion protocol only 7.5% of the tumours could be biotinylated successfully

at least in a significant part of the cross-section (Fig. 3.3).

We improved the perfusion of tumours in mice by introducing several

modifications to the perfusion protocol.

In a first attempt (see 3.1.1), we increased the perfusion flow rate accounting

for the fact that the heart minute volume in the living mouse is much higher

(11-36 ml/min) than the perfusion rate in the original in vivo biotinylation

protocol (~1.5 ml/min). We therefore concluded that, under these conditions,

the perfusion solution may follow the vascular structure of least resistance

and avoid passing through the tumour vessels. However, the increase of the

flow rate did not lead to a better perfusion of tumours (Text 3.1.1 and Fig.

3.1). The histochemical analysis of heart, kidney and liver, which showed a

staining not only of defined vascular structures as in experiments with low flow

but also staining deeper in the tissue, indicate that vessels may break during

these high flow perfusions. This might also explain that not only tumour but

also muscle tissue was not biotinylated and that the perfusion solution

accumulated in the abdomen, which became extremely swollen. Rather than

flowing through the microvasculature of tumour and muscle, the perfusion

solution may leak through broken vessels under these conditions. Thus,

perfusions were continued to be performed with a low flow rate (< 2ml/min).

In the live animal, the blood is able to reach the tumour, because cancer cells

needs supply of oxygen and nutrients for growing. We therefore compared the

accessibility of vascular structures for Bisbenzimide H33342 (Hoechst dye), a

nuclei-staining fluorescent dye, either by intravenous injection or by perfusion.

From these experiments we learned that the tumour is perfused less

efficiently with blood than the normal organs and that by omission of the PBS

wash step prior to in vivo biotinylation. More microvascular structures are

reached, at least in normal organs. We concluded that the vessels become

less accessible for perfusion the more time elapses by after the opening of the

149

thorax. From this time, the mouse is dying and the physiological nature of the

vessels probably changes. These findings convinced us to omit the PBS wash

in our perfusion procedure In principle, the omission of a work step might lead

to the artificial recovery of biotinylated blood proteins in the proteomic

analysis, as this was indeed the case for many blood proteins identified in the

gel-free proteomic analysis (unpublished data from Giuliano Elia). A gel-free

proteomic analysis of biotinylated proteins from perfusion experiments

according to the original protocol was not performed yet, but would be

interesting to evaluate the influence of the washing step on the recovery of

blood proteins in the sample.

Before testing the omission of the PBS wash step in further in vivo

biotinylation experiments, we took two other aspects into account. We

reasoned that the low accessibility of tumours for the in vivo biotinylation

might also be a result of a collapse of microvessels due to a too low oncotic

pressure of the perfusion solutions. An influx of water from the perfusion

solution into the tissue might occur, leading to a compression of the

microvessels. Thus, 21 tumour-bearing mice were submitted to in vivo

biotinylation applying the original protocol but without the previous PBS wash

and with 10% dextran 40 in the biotinylation and in the quenching solution

(see Tab. 2.1). Dextran 40 is a ~40 kDa polymer which binds water molecules

and therefore increases the oncotic pressure.

Furthermore, we reasoned that a cold environment (as well as the injury

during surgery) may possibly lead to a circulatory shock causing a

vasoconstriction in the periphery of the body in order to maintain the function

of the more important inner organs. Therefore, additional 7 mice were

perfused according to the final optimized in vivo biotinylation protocol

specified in 2.2.2 featuring the omission of the PBS wash prior to the

biotinylation step, the pre-warming of the perfusion solutions to 38°C, the

warming of the animal during anesthesia and perfusion using a heating pad

and an infrared lamp, as well as the presence of 10 % (w/v) Dextran 40 in the

perfusion solutions.

150

The result of a preliminary comparative statistic (Fig. 3.2) showed that only a

small portion of tumours were biotinylated at least in a significant part (Fig.

3.2, categories C + D) when the original perfusion protocol was used (7.5%).

By omission of the PBS wash step and using dextran in the perfusion

solutions this rate could be increased significantly (24%). Even though the

number of animals submitted to the final optimized in vivo biotinylation

protocol (n=7) is too low to draw definite conclusions, it seems that omission

of the PBS wash, addition of dextran to the perfusion solutions, pre-warming

of the perfusion solutions to 38°C and warming of the animal during

anesthesia and perfusion even lead to a much higher proportion of

successfully biotinylated tumours (43%).

The perfusion of the tumour vasculature continues to be less efficient than the

one of healthy organs, not only in subcutaneous tumour vessels, but also

when we used renal cell carcinoma (RENCA) tumours as an orthotopic model

in balb/c mice kidneys (unpublished data from JAscha Rybak and Giuliano

Elia; RENCA-bearing mice kindly provided by Raffaella Giavazzi (Bergamo,

Italy)). Why are tumours less accessible for perfusion than normal tissues? In

the living animal, blood obviously must have access to the tumour to enable

oxygen and nutrient supply. However, the tumour vessels are immature and

differ from their normal counterparts: architecturally, they are irregularly

shaped, dilated and tortuous, and even contain dead ends [Konerding et al,

2001]. Extensive fenestration, an abnormal basement membrane and

unusually wide gaps between adjacent endothelial cells make them leaky

[Roberts et al., 1997; Jain et al., 1987; Hashizume et al., 2000]. These

immature vessels are apparently more susceptible to blood clogging, collapse

or disrupture. Intravital microscopy studies have revealed that the tumour

vasculature shows a dynamic pattern of opened and closed vessels [Vajkoczy

P., oral communication]. Padeira, Jain and coworkers point out that interstitial

fluid pressure, which is characteristically elevated in solid tumours, is about

equal to the microvascular pressure in tumours and therefore unlikely to

mediate the collapse of perfused vessels (Padera et al., NATURE 2004).

Furthermore, these authors show evidence that, in a growing tumour, the

151

proliferating cancer cells itself, cause intratumour vessels to compress and

collapse (Padera et al., NATURE 2004).

The perfusion of rodents with paraformaldehyde, e.g., is a common method

for the fixation of different tissues including tumours. Donald McDonald and

coworkers used intravital perfusion of tumour-bearing mice with different

lectins for the characterization of the tumour microvasculature [e.g., Debbage

et al., J Histochem Cytochem 1998]. The authors use a slightly different

perfusion protocol [Hashizume et al., 2000]: they insert the perfusion cannula

through the left heart ventricle directly into the ascending aorta (which we

were not able to reproduce), perform the perfusion with a pressure of 120-140

mm Hg (which remains to be tested for our application) and claimed that, by

this, vasculature in the tumour was free of blood and preserved in an open

state. Furthermore, McDonald’s confirms the role of the temperature and the

oncotic pressure of the perfusion solution [McDonald, oral communication].

They proposed the addition of the plasma expander poly vinyl pyrrolidone to

the perfusion solutions and pointed out, that the time frame between opening

of the thorax (when the mouse begins to dye) until starting the perfusion has

to be reduced to a minimum, at least < 30 sec. During this time, the heart has

to be cannulated, which took us usually about 1 – 2 min in the beginning. In

later experiments we achieved to reduce this time to ~40 -60 sec.

It is interesting that McDonald and co-workers only in earlier work [Thurston et

al., 1996] applied the lectin indeed via perfusion, later they changed protocol

and injected the lectin intraveneously in the still living animal prior to the

perfusion with fixative [Ezaki et al., 2001]. As also suggested by the results we

obtained by injection or perfusion of tumour-bearing mice with Hoechst dye,

the application of the labelling reagent into the still living animal might lead to

a better labelling in tumours. However, this is not directly transferable to the in

vivo biotinylation, as the blood contains so much primary amino groups, that it

is not possible to administer high enough doses of biotinylation reagent to

overcome this quenching and to have enough excessive reagent to label

vascular structures.

152

We now routinely reach a tumour perfusion rate of almost 50% with the final

optimized protocol. Enough tumours can be successfully biotinylated to

perform comparative proteomic analyses of the accessible proteins. This is

shown by the results of the gel-free proteomic shot-gun analysis performed by

Giulino Elia (unbublished data) which led to hundreds of protein identifications

in the affinity purified tumour samples from in vivo biotinylation experiments.

4.2 Gel-based proteomic analysis For the in vivo biotinylation technology, besides the perfusion of tumours, one

of the major challenges is the comparative proteomic analysis of the labelled

proteins. For the separation of labelled proteins, affinity capturing on

streptavidin was chosen. However, the release of biotinylated proteins from

streptavidin resins remained a major problem, due to the extraordinary

stability of this complex. Rybak and coworkers developed an optimized

protocol for the quantitative elution of biotinylated proteins from streptavidin

sepharose, featuring harsh elution conditions and competition with free biotin

[Rybak et al., 2004]. Using this method we achieved the quantitative recovery

of biotinylated proteins from organ homogenates, obtained from mice

perfused with Sulfo-NHS-LC-biotin (Fig 3.5). However, due to the high

concentration of salts and detergents in the elution buffer, the direct

processing of the samples by proteolytic enzymes and by mass-spectrometric

analysis is not feasible. The apparently only possible subsequent technique is

1D-SDS-PAGE, which, consequently, was utilized for the analysis of eluted

biotinylated proteins from in vivo biotinylation (Fig. 3.4 and 3.5). In this

analysis we faced the problem that the protein concentration in the eluted

sample is so low that only few protein bands are detectable in the gel. Out of

these, only the few strongest bands gave positive identifications in the mass

spectrometry analysis. The volume which can be loaded on a gel is limited.

Thus, only a higher concentration of proteins in the eluate could lead to more

and more intense gel bands. Our approach to concentrate the proteins on the

resin by applying more input protein extract and less streptavidin sepharose

153

and elution solution failed due to the limiting capacity of the resin under these

conditions (Fig. 3.6.). Using more streptavidin sepharose, however, would in

turn force us to use more elution buffer to assure quantitative elution of the

biotinylated proteins. The other possibility was the concentration of the eluted

samples itself, which we tested using spin dialysis (Fig. 3.7). The high

concentration of salts and SDS in the elution buffer, however, led to

precipitation when samples were concentrated more than ~5 x (see Fig. 3.7).

The gel-based proteomic analysis indeed led to the first identification of some

proteins labelled in the in vivo biotinylation (Fig. 3.5), among which the kidney-

specific cadherin 16 was found to be the most abundant accessible protein in

rodent kidneys.

However, only few protein bands were at all visible in the gel and only the rare

intense bands led to protein identification. In summary, the gel-based

proteomic analysis suffers from poor sensitivity.

4.3 Gel-free proteomic analysis As the gel-based analysis of biotinylated proteins recovered from in vivo

biotinylation experiments only led to few identifications, a gel-free proteomic

approach had been set up in our laboratory by Jascha Rybak and Giuliano

Elia featuring an affinity capture of biotinylated proteins on streptavidin

sepharose, a tryptic digestion of the proteins directly on the resin (“on-resin

digestion”) and a mass spectrometry analysis.

Using this technology, a significantly higher number of proteins could be

identified compared to the previous gel-based proteomic analysis. Elia et al.

identified up to more than hundred proteins labelled in different organs during

in vivo biotinylation [Rybak et al., manuscript in preparation].

154

In theory, this shot-gun approach is not quantitative, as it can only show the

presence or absence of a protein. The statistical frequency of occurrence of a

certain peptide in an analysis, however, may indicate the abundance of the

corresponding protein in the biotinylated fraction. Furthermore, this technology

is thought to be combined at a later stage with the two dimensional peptide

mapping technology invented in our laboratory by Simone Scheurer and

Christoph Rösli [Scheurer et al., manuscript submitted]. (This technology

allows the relative quantification of peptides from digested biotinylated

proteins by separation with high-pressure liquid chromatography and

subsequent mass spectrometry analysis yielding a two dimensional peptide

map (first dimension = elution from HPLC, second dimension = molecular

weight according to time of flight analysis).)

A reduction of sample complexity by pre-fractionation of the protein extracts

was thought to further improve the gel-free proteomic analysis and to lead to

more protein identifications by mass spectrometry. Therefore, a

hydroxyapatite-based chromatography method for the pre-fractionation of the

protein extracts from mice perfused with a biotinylation reagen was set up in

the scope of this diploma thesis (see 4.4).

4.4 Prefractionation by hydroxyapatite chromatography As starting conditions a protocol from Simone Scheurer et al. (unpublished

data from our laboratory) was used. In preliminary experiments, the

fractionation of a standard protein mixture used to find optimal separation

conditions (see 3.3). The optimized conditions included the total omission of

phosphate in the first elution buffer (buffer A) in contrast to protocols in

literature [Ichimura et al., 1994, Dong et al.1996]. Different pH values of the

elution buffers did not lead to significant changes of the elution profile, even

though a strong influence of the pH was reported in literature [Tetsuro and

Tsuneo, 1986].

155

The designed protocol was successfully applied to the separation of a

complex kidney protein extract (Fig. 3.14), which led to further optimisations

(omission of the calcium chloride, variation of the gradient type, fraction size

and phosphate concentration in elution buffer B).The optimised protocol was

applied for the fractionation of a protein extract from in vivo biotinylated renal

cell carcinoma (RENCA) tumours. Different signal patterns in the lanes of the

various fractions in the SA blot analysis of the fractions of this sample

indicated the successful fractionation of the biotinylated proteins. The

fractions of this successfully pre-fractionated sample were submitted to a shut

gun proteomic analysis performed by Giuliano Elia.

Virtually, each protein was identified mostly in one single fraction indicating a

successful fractionation of such a complex protein mixture by hydroxyapatite

chromatography.

Exceptions were trypsin and albumin which are nearly present in all fractions.

These highly abundant proteins were probably artefacts from the perfusion

procedure as albumin is one of the most abundant blood proteins and trypsin

is used for the on resin digestion.

Different proteins were identified in the fractions of the chromatography

compared to the unprocessed sample (see table 3.1).

In the shut gun proteomic analysis, the number of identified proteins was not

significantly enhanced compared to the analysis of the non-fractionated

protein extract. This could be explained either with technical problems with the

column performance which led to a missing protein identifications in fraction

16 to 21/22 or with the extremely low protein amount which was present in the

single fractions. An increase of the protein amount loaded on the HA catridge

would lead to a higher amount of protein in the various fractions, and

therefore might lead to a higher number of protein identifications. However,

other HA columns with a higher loading capacity would be needed.

Overall, pre-fractionation by hydroxyapatite chromatography seems to be a

promising tool for the proteomic analysis of biotinylated proteins.

156

4.5 Alternative perfusion reagents In the gel-free proteomic analysis of biotinylated proteins from protein extracts

from mice perfused with sulfo-NHS-LC-biotin, in addtion to the expected

proteins from cell plasma membrane and extracellular matrix, several

intracellular proteins were identified (see 3.3). This was unexpected

considering that the charged sulfate-group should prevent the biotinylation

reagent from penetrating biological membranes. To a minor certain extent,

cell damage during the perfusion procedure may lead to the release of inner

cell compartments and proteins. However, these released and biotinylated

proteins would be washed away at latest by the last perfusion step with

quenching solution.

Peirce and co-workers [Peirce at al.,2004] showed that the cell surface

biotinylation of permeabilized cells with sulfo-NHS-LC-biotin led in a

significant proportion to biotinylation of the whole intracellular compartement,

while this was not the case for sulfo-NHS-SS-biotin. Thus, we reasoned that

the disulfide bond of sulfo-NHS-SS-biotin might be cleaved when entering the

cell due to the reducing intracellular conditions. Therefore, we decided to test

in vivo biotinylation using sulfo-NHS-SS-biotin as biotinylation reagent.

In a pre experiment, we perfused Black six mice with sulfo-NHS-SS-biotin.

Histochemical analysis gave evidence that in vivo biotinylation with this

alternative biotinylation reagent in principle is feasible (data not shown).

Therefore, F9 tumour-bearing SvEv/129 mice were submitted to in vivo

biotinylation with sulfo-NHS-SS-biotin according to the optimized protocol (see

2.2.2). Overall, the subsequent SA blot and histochemical analyses showed

low intense signals compared to the perfusion experiments with sulfo-NHS-

LC-biotin. This suggests that both proteins (and other structures exhibiting

primary amino groups) were biotinylated to a lesser extent, or the bound biotin

was cleaved during the further procedure. This might be due to a cleavage of

the disulfide bond either before or after the reaction with accessible

structures.

157

Protein extracts of all kidney, liver, muscle and tumour from mice perfused

with sulfo-NHS-SS-biotin whose tumour sections indicated a positive result in

the histochemical analysis were submitted to a shot gun proteomic anaylsis.

In this analysis, fewer proteins could be identified compared to previous shot

gun proteomic analysis of protein extracts from mice perfused with sulfo-NHS-

LC-biotin. This confirms the conclusion drawn from SA blot and histochemical

analysis that fewer proteins were biotinylated with the sulfo-NHS-SS-biotin.

However, a reduction of the proportion of cytosolic proteins was not found.

Therefore, the decrease of the amount of biotinylated proteins has to be

explained by the lower stability of the sulfo-NHS-SS-biotin compared to the

sulfo-NHS-LC-biotin.

A more detailed analysis of the data obtained in the gel-free proteomic

analysis may reveal whether different proteins are labelled by the two

reagents and therefore might be used complementarily. However,

preliminarily we conclude that sulfo-NHS-LC-biotin is the superior reagent for

in vivo biotinylation.

Another strategy was approached to tackle the problem of unwanted labelling

of cytoplasmatic proteins during in vivo biotinylation. High molecular weight

polymers should not be able to diffuse through biological membranes. Thus, a

dextran-NHS-derivative was synthesized, providing the following features: a)

biotin residues for affinity capturing on streptavidin, b) NHS groups for the

reaction with primary amines c) a fluorescent label for the direct detection by

fluorescent microscopy of the corresponding organ sections or the direct

detection of protein gel bands in a fluorescence imager, and d) such a high

molecular weight that it should not be able to penetrate biological membranes.

Therefore, a commercially available 70 kDa dextran modified with 5 lysines

and 40 biotins was reacted with a 100 x excess of disuccinimidyl tartrate

(DST), which acts as crosslinker of primary amino groups. After the synthesis

reaction, DST in excess had to be eliminated.

We established a procedure for the elimination of the low molecular weight

compound from the high molecular weight compound in the dextran

modification reaction.

158

159

Subsequently, we performed the activation of biotinylated dextran, using

bifunctional cross-linker DST.

Due to time restrictions, the characterization of the dextran-biotin-NHS-

derivative, and the optimisation of the synthesis reaction for dextran-NHS-

biotin derivaive was not performed. Thus, the usefulness of this type of

biotinylation reagents for the in vivo biotinylation technology remains to be

evaluated.

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6 CURRICULUM VITAE Name: Stefanie Pfaffen

Born: 29.05.1980 in Visp (VS)

Citizen of: Mund / VS (CH)

Address: Stale

3922 Stalden (VS)

Education 1987-1993 primary school in Stalden

1993-1995 secondary school in Stalden

1995-2000 Kollegium “Spiritus Sanctus” in Brig

2000 Matura, Typus B

2000-2004 Studies in pharmaceutical sciences at the Swiss

Federal Institute of Technology, Zürich (ETHZ)

2003 Diploma thesis “Optimization of the in vivo

biotinylation technology for the discovery of

accessible tissue-specific markers“ in the laboratory

of Prof. Dr. Neri at the Department of Chemistry

and Applied Biosciences of the ETH

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