Surface Modified Capillaries in Capillary Electrophoresis ...173240/FULLTEXT01.pdf · laser desorption/ionisation mass spectrometry of intact and ... 2 Lys Tag labelling for enhanced

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2009

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 603

Surface Modified Capillaries inCapillary Electrophoresis Coupledto Mass Spectrometry

Method Development and Exploration of thePotential of Capillary Electrophoresis as a ProteomicTool

AIDA ZUBEROVIC

ISSN 1651-6214ISBN 978-91-554-7411-9urn:nbn:se:uu:diva-9554

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“Real success is finding your lifework in the work that you love”

David McCullough

To Ishak

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Hardenborg, E., Zuberovic, A., Ullsten, S., Söderberg, L., Hel-

din, E., Markides, K. E. Novel polyamine coating providing non-covalent deactivation and reversed electroosmotic flow of fused-silica capillaries for capillary electrophoresis. Journal of Chromatography A, (2003) 1003, 217–221.

II Ullsten, S., Zuberovic, A., Wetterhall, M., Hardenborg, E.,

Markides, K. E., Bergquist, J. A polyamine coating for en-hanced capillary electrophoresis electrospray ionization-mass spectrometry of proteins and peptides. Electrophoresis, (2004) 25, 2090-2099.

III Zuberovic, A., Ullsten, S., Hellman, U., Markides, K. E., Ber-

gquist, J. Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries. Rapid Communications in Mass Spectrometry, (2004) 18, 2946-2952.

IV Zuberovic, A., Hanrieder, J., Bergquist, J. and Wetterhall, M.

Proteome profiling of human cerebrospinal fluid: exploring the potential of capillary electrophoresis with surface modi-fied capillaries for analysis of complex biological samples. European Journal of Mass Spectrometry, (2008) 14, 249-260.

V Hanrieder, J., Zuberovic, A. and Bergquist, J. Surface modi-

fied capillary electrophoresis combined with in solution IEF and MALDI-TOF/TOF mass spectrometry: A miniaturized, gel-free multidimensional electrophoresis approach for pro-teomic profiling of complex human body fluids - exemplified on human follicular fluid, Journal of Chromatography A, (2009), In Press.

VI Zuberovic, A., Hanrieder J., Wetterhall, M. and Bergquist J. CE MALDI TOF/TOF MS for multiplexed quantification of proteins in human ventricular CSF. Electrophoresis, Under revision.

Reprints were made with kind permission from the publishers. Other papers not included in the thesis: - Ullsten, S., Zuberovic, A. Bergquist,, J. Adsorbed cationic poly-

mer coatings for enhanced capillary electrophoresis/mass spec-trometry of proteins. Capillary Electrophoresis – Methods and Protocols (2008), Chapter 25.

- Ramström M., Zuberovic, A., Grönwall, C., Hanrieder, J. Bergquist,

J., Hober, S. Development of affinity columns for the removal of high-abundant proteins in cerebrospinal fluid. Biotechnology and Applied Biochemistry, (2009) 52, 159-166.

- Wetterhall, M., Zuberovic, A., Hanrieder, J. Bergquist, J. A shot-

gun proteomic study of immunoaffinity depletion spin columns for the removal of multiple high-abundance proteins in human cerebrospinal fluid. In manuscript.

Author’s contribution to the papers:

Paper I – Performed experimental work, except for the synthesis of PolyE-323. Took part in the discussion of the results and the writing of the paper. Paper II – Planned and performed all CE-MS experiments in collaboration with Sara Ullsten and Magnus Wetterhall. Took part in the discussion of the results and writing of the paper. Paper III – Planned and performed all experiments in collaboration with Sara Ullsten and Ulf Hellman. Wrote the paper together with Sara Ullsten. Paper IV – Planned and performed the experimental work. Performed MS experiments in collaboration with Jörg Hanrieder. Wrote the paper. Paper V – Planned and performed the experimental work and wrote the paper together with Jörg Hanrieder. Paper VI – Planned and performed the experimental work. Performed MS experiments in collaboration with Jörg Hanrieder.Wrote the paper. The projects were conducted under supervision of Jonas Bergquist, Papers I-VI, Karin Markides, Paper I-II, and co-supervision of Magnus Wetterhall in Papers IV and VI.

Contents

1 Introduction................................................................................................11

2 Bioanalysis.................................................................................................13 2.1 Biological samples and sample handling ...........................................14

3 Capillary electrophoresis (CE)...................................................................16 3. 1 Basic theory of CE ............................................................................16

3. 1. 1 CE instrumentation ...................................................................16 3. 1. 2 Electrically driven separation in CE .........................................17

4 Capillary modifications..............................................................................19 4. 1 Protein-wall interactions....................................................................20 4. 2 Cationic coatings ...............................................................................20

5 Coupling CE to mass spectrometry (CE-MS)............................................24 5. 1 CE-ESI-MS .......................................................................................25 5. 2 CE-MALDI-MS ................................................................................27

6 Sample preparation ....................................................................................30 6. 1 Sample fractionation methods ...........................................................32

6. 1. 1 Immunoaffinity depletion of high-abundant proteins ...............32 6. 1. 2 In solution isoelectric focusing .................................................33

6. 2 Chemical labelling.............................................................................34 6. 2. 1 Multiplexed quantitative proteomic analysis with mass spectrometry using stable isotope labelling .........................................34 6. 2. 2 Lys Tag labelling for enhanced protein identification in MALDI-MS .........................................................................................36

7 Proteomic analysis .....................................................................................37 7. 1 Analysis of proteomic samples with CE ...........................................38 7. 2 Analysis of human body fluids..........................................................39

7. 2. 1 Cerebrospinal fluid ...................................................................40 7. 2. 2 Human follicular fluid ..............................................................42

8 Concluding remarks and future aspects .....................................................43

9 Summary in Swedish .................................................................................45

Ytmodifierade kapillärer för kapillärelektrofores kopplad till masspektrometri .......................................................................................45

Metodutveckling och undersökning av kapillärelektroforesens potential som ett proteomikverktyg ....................................................................45 Kapillärelektroforetisk separation av proteiner och peptider...............45 Masspektrometri ..................................................................................46 Provupparbetning.................................................................................46 Parallell kvantifiering av proteiner med MS........................................47

10 Acknowledgements..................................................................................48

References.....................................................................................................50

Abbreviations

APS 3-aminopropyltrietoxysilane BGE Background electrolyte CE Capillary electrophoresis CEC Capillary electrochromatography cIEF Capillary isoelectric focusing CNS Central nervous system CSF Cerebrospinal fluid CZE Capillary zone electrophoresis Da Dalton EOF Electroosmotic flow ESI Electrospray ionisation hFF Human follicular fluid HPLC High performance liquid chromatography HV High voltage iTRAQTM Isobaric tag for relative and absolute protein quantification IVF In vitro fertilization Lys Tag 2-methoxy-4,5-dihydro-1H-imidazole MALDI Matrix-assisted laser desorption/ionisation MAPTAC [3-(methacryloylamino)propyl]trimethylammonium cloride MEKC Micellar electrokinetic chromatography MS Mass spectrometry MS/MS Tandem mass spectrometry 2D PAGE Two-dimensional polyacrylamide gel electrophoresis pI Isoelectric point RSD Relative standard deviation TBI Traumatic brain injury TOF Time-of-flight UV Ultraviolet vCSF Ventricular cerebrospinal fluid

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1 Introduction

The will and the interest to better understand ourselves and our surrounding environment is the driving force in research. We want to be able to regulate and control the functions and processes of life. In order to explore these processes, development of new methods for measuring components in different samples in a fast and reliable way is needed. A current trend in life science is the development and use of various types of "omics" (proteomics, genomics, metabolomics, etc.) which include analysing complex samples containing proteins, DNA or product substances from metabolic pathways. To be more precise, proteomics considers a comprehensive analysis including identification, characterization and quantification of all proteins expressed in the cell, the tissue or the organism at a certain time point1. Proteins, together with peptides, are responsible for a wide variety of functions in the body, and because of that, they possess the widest inherent chemical diversity of properties. Consequently, their complexity provides a challenge for the analytical techniques that are used to explore them.

Capillary electrophoresis (CE) is a separation technique in which the analytes are separated on the basis of differences in their charge-to-size ratios. This makes CE especially suitable for the analysis of molecules with a broad range of sizes, charge and hydrophobicity, as proteins and peptides. Another attractive feature of CE is its ability to handle minute sample amounts, with nano-litre injection volumes, which makes it ideal for applications when the sample volumes are limited, as often is the case in analysis of body fluids2-4, single cells5,6 and other small volume analysis of biofluids7. However, when using CE for separation of proteins and peptides their basicity causes adsorption to the capillary inner wall and deteriorates the separation. The adsorption has thus been a problem to address in order to use advantageous properties of the CE technique. In addition, the small injection volume has often been considered as a drawback of the CE technique in analysis of complex biological samples, as well as the nature of its injection has represented a reproducibility problem in quantitative chemical analysis.

Mass spectrometry (MS), is today one of the most advanced detection techniques. It yields maximum accurate information in short time, consuming minimal amounts of sample. MS provides robustness and reproducibility of analysis as well as high throughput possibilities via automatic data acquisition. When combined with soft ionisation techniques,

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like electrospray ionisation (ESI)8 and matrix-assisted laser desorption/ionisation (MALDI)9,10, MS allows simultaneous analysis of proteins and peptides with high resolution. However, in order to reach ultimate mass sensitivity a separation step prior to MS detection is often needed11-13. The possibility of combining the properties of MS with a fast and high-efficiency separation provided by CE to meet the analytical challenges, that the complexity of biological samples represents, has been recognized for about two decades14. Nevertheless, the previously mentioned drawbacks of CE together with a number of other challenges including huge dynamic range of analyte concentrations in biological samples, interfacing strategies and incomplete ionisation efficiency in MS have prevented CE to reach a wider use in bioanalysis and proteomics.

The aim of this thesis is to describe and discuss development of CE-MS methods to minimise or circumvent the above outlined challenges in analysis of proteins and peptides in complex biological samples. The thesis is based on six papers. Papers I-III focus on the method development of surface modified capillaries and interfacing of CE to MS via ESI and MALDI. Papers IV-VI deal with sample preparation strategies in new approaches of applying the developed CE-MS methods in proteomic analysis. The thesis provides overviews of the covered research areas and aims to put the obtained results in the papers into a global context.

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2 Bioanalysis

As a short overview of the field of bioanalysis as it has been performed during the last century this chapter is going to give an introduction to the bioanalysis in modern time by identifying the trends and challenges in the development of new analytical techniques. Through the description of the work presented in the rest of this thesis it will eventually lead into the field of proteomics in chapter 7 and describe how the new possibilities of the developed approaches can serve to meet the existing analytical difficulties in this field. The research terminology of today is dominated by bio-words among which bioanalysis is one of the most frequently used. The term associates to the most recent trends in the research although it has been the driving force in analytical chemistry for a long time. The beginning can perhaps be traced back to the building of expensive and hard to operate analytical systems like the ultracentrifuge15 and instrumentation for moving-boundary electrophoresis16 at the time when the proteins were classified as colloids. These techniques, the finest analytical tools available in those days, enabled for the first time precise determination of molecular weights (Mw) by forcing the proteins to sediment with the aid of high centrifugal forces and to perform accurate measurements of absolute mobilities of proteins respectively. Through almost a century of bioanalysis, the way to the nanotechnology of today has passed by the times of many bioanalytical techniques that have made more or less impression in history. With or without intention, the development of bioanalytical devices has had a tendency towards miniaturisation. The expensive and complicated gadgets were successively replaced by other methods starting from: size-based fractionation of proteins and peptides on dextran gels17 via polyacrylamid gel electrophoresis (PAGE)18, isoelectric focusing (IEF)19, sodium dodecyl sulfate (SDS)20, capillary zone electrophoresis (CZE)21 and Sephadex22 to isotachophoresis (ITP)23 and two dimensional gel electrophoresis (SDS-PAGE)24,25. After the invention of ESI26 and MALDI9,10, at the very end of the eighties, the bioanalytical instrumentation was finally crowned with mass spectrometry. Combined with 2-D gel protein mapping MS became then a major tool for biology, biomedicine and bioanalysis which has made this technique to the main contributor in the proteomics growth during the last decade.

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The desire to understand the detailed mechanisms that control the dynamics of cellular events, which occur at the nanoscale, is a central issue in current life science research. In clinical proteomic studies, in particular, there is a great interest in identifying proteins that can serve as biomarkers for human diseases. It seems likely that many of the potential biomarker proteins are present in the human body at very low levels. Consequently, their identification is a formidable analytical task. In order to overcome the challenges that the cellular environment represents and the inherent restrictions of the bioanalytical approaches existing today the ongoing development of smaller, faster and more compact bioanalytical devices has unconditionally to proceed.

2.1 Biological samples and sample handling Samples of biological origin are often very complex in the context that they contain many different types and sizes of components in a wide range of concentrations. In addition to the complexity they also have a very dynamic nature, which means that the compositions of the sample can vary drastically between sampling occasions. Human body fluids do not only transport nutrients and contain metabolic end products, they also serve as media for substances that hold a lot of information. Since they are in direct contact with the extracellular fluid in organs their composition can be used as a gauge for the wellbeing of an organism. Human body fluids are therefore of great importance as sources of information in e.g. clinical diagnosis, prognosis, drug development and understanding of physiological processes in the human body.

Analysis of biological samples involves many challenges concerning potential sample loss and thereby loss of important information as well as reproducibility. A direct analysis of unprocessed sample would ideally be performed, which would in most cases prevent all artificial losses and biased correlation of findings that can arise from sample preparation. Unfortunately, it is often necessary to expose biological samples to some kind of sample pre-treatment in order to minimise their complexity and reduce the dynamic range of concentrations. This need is especially emphasised in MS contexts where it is important to prevent contamination of the mass spectrometer with, for instance, salts, lipids or carbohydrates, if proteins and peptides are analysed. It has been reported that an analyte in a sample extracted from a tissue, may produce much lower signal (10-100 times lower) than the identical amount of its synthetic counterpart in a standard solution27. Difficulties with biological sample handling can arise from the sampling occasion28 and the storage29 through all the steps of sample preparation and during the whole process of analysis. Significant sample loss, often defined as adsorption, might also be conceived in the plastic ware used for the

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analysis like pipette tips, sample tubes, columns for desalting and concentration and sample transfers. In protein/peptide analysis sample tubes from different batches have been experienced to introduce big differences in mass spectra. The adsorption could also increase with decreasing sample amounts and with increasing sample purity27. It is therefore important to handle biological samples in a proper way in order to prevent loss of valuable information.

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3 Capillary electrophoresis (CE)

3. 1 Basic theory of CE Separation by electrophoresis is based on differential movement of charged species (ions) by attraction or repulsion in an electric field21,30. In CE, electrophoretic separation is performed in capillaries made of fused silica (a metal free glass material), typically 25-75 μm in inner diameter, which are usually filled with a buffer. The high electrical resistance of the capillary enables the application of very high electrical fields (100-500 V/cm) with only minimal heat generation. The use of high electrical fields results in short analysis times and high peak efficiency and resolution. Other properties that characterise CE are the minimal sample volume requirements (low nanolitre range) and its diversity of application. Since the introduction of CE in the early 1980's, it has proved useful for separation of a wide range of compounds starting from small inorganic ions, amino-and organic acids via peptides, proteins and DNA fragments to whole cells and virus particles31. There are also other electromigration methods in capillary formats like capillary electrochromatography (CEC), micellar electrokinetic chromatography (MEKC) and capillary isoelectric focusing (cIEF) that have been developed and are intensively used in similar manners to classical capillary zone electrophoresis (CZE, another frequently used abbreviation of the CE technique) for different analytical purposes32,33,34.

3. 1. 1 CE instrumentation One of the key features of CE is the simplicity of the instrumentation. A CE system consists of a buffer-filled fused silica capillary, where the separation takes place, two buffer reservoirs connected to a high voltage power supply using platinum electrodes and a detector, usually placed closer to the outlet buffer reservoir, connected to a computer for data collection. A basic instrumental set-up of CE is shown in Figure 1.

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Figure 1. Schematic picture of the instrumental set-up for capillary electrophoresis.

Sample is loaded into the capillary on the opposite side of the detector. Upon application of a potential difference over the capillary (usually 15-30 kV) the separation is started. Ultraviolet (UV) detectors are most commonly used, but numerous other detection techniques can be employed. Mass spectrometry is, however, the most universal and presently unrivalled detection technique that is the best choice in combination with separation in the same system. In this thesis CE is interfaced to MS using on-line electrospray ionisation, Paper II, and off-line MALDI ionisation, Paper III -VI.

3. 1. 2 Electrically driven separation in CE The mechanisms responsible for separation in CE are different from those in chromatography. In CE there is a specific electrophoretic phenomenon called electroosmotic flow (EOF) which simply can be considered as an electric pump that continuously pushes the solvent inside the capillary towards the detector. This happens when the inner wall surface of the silica capillary, consisting of protolytic silanol groups (Si-OH, pKa ~3-9), becomes activated35. The activation of the wall starts with the deprotonation of silanol groups (to anionic form, Si-O-) when solvents with pH > 3 are used, giving a negatively charged surface. The hydrated cations from the solvent start to accumulate near the surface to maintain charge balance forming an electric double layer at the inner wall of the capillary (Figure 2).

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Figure 2. Schematic figure of the electric double layer. ψ0, the surface potential; ψδ, stern layer potential; ζ, zeta potential. Arrows indicate decreasing of the potential ψ at distance x from the surface.

When the voltage is applied across the capillary the cations are attracted towards the cathode and start to move in that direction. Because they are solvated their movement drags the bulk solution in the capillary towards the cathode, giving rise to formation of the EOF. The EOF causes movement of nearly all species, regardless of charge, in the same direction. Thus, cations, neutrals and anions can be separated in a single run if the magnitude of the EOF is greater than the electrophoretic mobilities of anions in opposite direction. This process is depicted in Figure 3. Besides the different types of charges, other properties of analytes like their size and their net charge give rise to the differences in their migration times at a given pH of the BGE (background electrolyte) and the voltage applied over the capillary. In this way analytes achieve different velocities through the capillary and separate from each other.

Figure 3. Development of the electroosmotic flow. (A) Hydrated cations accumulating near to negatively charged fused silica surface (Si-O-). (B) Bulk flow towards the cathode upon application of an electric field.

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4 Capillary modifications

Modification of the inner wall in fused silica capillaries can be advantageous for various reasons. Different types of applications require different properties of the EOF or the separation. For instance, analyte-wall interactions can be prevented, as shown in Paper I. In addition, modifications of the EOF can increase the speed of analysis, improve reproducibility and resolution or increase compatibility with MS detection. Various strategies, that have been more or less successful, have been introduced to modify capillary walls. Using extremes of pH, that give highly negatively or positively charged capillary wall and repel analytes with the same overall polarity, has perhaps been the most trivial way to minimise analyte-wall interactions36,37,38. High salt concentrations and buffer additives are also approaches that have been investigated for this purpose39,40,41. However, the most efficient way of addressing the adsorption problems has been to use various types of deactivating coatings to mask the anionic and hydrophobic nature of fused silica35,42-44.

Capillary coatings can generally be classified as dynamic, static (physically adsorbed) or covalent, based on the nature of the attachment of the coating to the inner wall surface. Dynamic coatings are commonly prepared by adding a coating agent, often a surfactant or a polymer, to the running buffer to maintain the coating on the capillary wall surface45,46. A severe problem when employing dynamic coatings in CE is their incompatibility with MS. The presence of such additives in the BGE deteriorates the ionisation process during electrospray ionisation and causes contamination of the mass spectrometer, which makes their use problematic in combined separation-MS47,48. To adsorb the modifier to the capillary wall permanently by physical adsorption and to fix the layer permanently by covalent bonding are therefore the preferred strategies when CE is coupled to MS. Covalent coatings have often longer lifetime and can give rapid separations, however, binding procedures (silanisations) consist usually of several consecutive steps which makes coating methods more time-consuming than desired and introduces problems with irreproducibility49. A severe limitation is also the instability of siloxane bonds (Si-O-C) at neutral and alkaline pH which restricts the use of these capillaries to acidic pH lower than 5-650. Compared to covalent coatings, the experimental procedures for production of physically adsorbed layers are easier to carry out and are thus often

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considered as more economical coating procedures. Regeneration of the capillaries is more straightforward and a stable EOF can be obtained after adsorption of strong polycations51,52.

4. 1 Protein-wall interactions The tendency of a protein to interact with a surface may be dependent on several factors such as charge density, net charge of the protein, charge distribution on the protein and electrostatic properties of the adsorbing surface. Proteins contain both positively and negatively charged functional groups. The positively charged residues are primarily responsible for the interaction of proteins with the negatively charged silanol groups on the capillary wall, causing protein adsorption. Such electrostatic interactions can be influenced by pH, as mentioned above. However, even if the net polarity of a large protein is the same as the wall, regions of opposite charge or hydrophobic domains may still exist that will result in binding51. Furthermore, extreme pH of the BGE can work well for CE-MS analysis of peptides but is denaturing for proteins and the use of pH to control analyte-wall interactions is often not the choice. Interactions between proteins and the capillary are sometimes reversible and can be observed as tailing peak shapes or irreproducible migration times, but most often they lead to sample loss, peak broadening, poor resolution and long migration times. For this reason manipulating the inner wall chemistry of fused silica capillary, as developed in Paper I and used in Paper II-VI, is essential for capillary electrophoretic separation of proteins. Modifying the inner capillary wall, to prevent protein adsorption, enables simultaneous manipulation of the separation characteristics and regulation of the EOF. For these reasons, the use of suitable types of coatings to meet the analytical needs is of great interest for proteomic studies using CE.

4. 2 Cationic coatings Many of the difficulties that could be associated with the separation of proteins using CE two decades ago are today circumvented thanks to amines that are included in many novel strategies to reduce the protein adsorption phenomenon in CE. This class comprises a large number of compounds starting from mono-, di-, tri-, tetra-, as well as oligo-, and polyamines53,54. These compounds have been used in different ways to shield the negatively charged silica surface during analysis of peptides and proteins either as dynamic, static or covalent coatings. Covering the inner capillary surface with these positively charged compounds a reversed electroosmotic flow is generated, compared to bare fused silica capillaries, as shown in Figure 4.

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Figure 4. Schematic picture of capillary electrophoresis with a polycationic surface modified capillary.

Many of these coatings are covalently bonded monomers, such as APS (3-aminopropyltrietoxysilane) and MAPTAC ([3-(methacryloylamino)propyl]- trimethylammonium cloride), suitable to generate high EOF and to give fast separations at low pH, the characteristics required in sheathless CE-ESI-MS of peptides49,55,56,57,58,59. Most monomers, however, fail to provide complete coverage of the silica surface, which may result in reverted direction of EOF, towards the cathode, particularly when a pH higher than 5-6 is used at which siloxane bonds often show hydrolytic instability60,61.

Attention is, thus, easily devoted to coating methods consisting of fast and simple rinsing procedures with polymers that give high surface coverage and that easily can be regenerated62-72. The use of physically adsorbed cationic polymer coatings has thus also been attractive for applications outside the CE world. In chip technology the interest for such coatings has increased since the miniaturisation of the separation systems emphasises the importance of controlling the surface properties. Furthermore, modifying the surface of microchannels enhances their applicability in various fields of microfluidics73-75. The concept of chip technology has also been to make fabrication of microfluidic devices less expensive, which makes development of adsorbed coatings particularly interesting. Also in such contexts, outside our group, the PolyE-323 surface has been synthesised and presented as a good alternative76. Despite the success of all deactivation methods developed until today there is no universal coating suitable for all

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types of applications. There has always been a need for continuous refinements of already existing or preparation of new surfaces with different properties in hydrophobicity, polarity, magnitude and direction of the EOF77,78. This ongoing development contributes with new possibilities and knowledge on how to produce new types of surfaces that are stable and uniform and that give high a surface coverage. Thus, the trend of today, to adapt the coating best suitable for a given sample, is presumably here to stay. For separation of biomolecules coatings with strong hydrophilic properties are usually most optimal, while hydrophobic properties may be required in other applications. Some of the most appreciated characteristics of a coating are high stability under the conditions required for separation and preferably over a broad pH range for the BGE44. In Paper I a novel polycationic polymer, PolyE-323, was synthesised and employed as an adsorbed coating for CE-separation of basic proteins. The structure of this polymer is shown in Figure 5.

Figure 5. Schematic structure of the PolyE-323 polymer.

PolyE-323 consists of short aliphatic blocks of three and two carbon atoms in length between each nitrogen atom, thereof the 323 in its name. It also contains a hydroxyl group that participates in immobilisation of the polymer onto the capillary wall by hydrogen bonding. A very important factor for the coating efficacy is the degree of freedom in the polymer backbone. A polymer of high rigidity fails to provide a smooth and complete coverage of the negatively charged silanol walls compared to a flexible polymer that can adjust its conformation on the surface. The short carbon chains not only contribute to increased flexibility of the polymer, but the tailored distance between the nitrogens produces a high cationic surface charge density which gives rise to generation of a high EOF. Physically adsorbed polymer coatings, such as PolyE-323, are simply prepared by flushing the capillary with an aqueous solution of the polymer, as described in Paper II. An electrostatically adsorbed layer is obtained that is very stable and gives a stable anodal EOF over a broad pH range as shown in Figure 6.

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Figure 6. Electroosmotic flow as a function of pH in fused silica capillary and a PolyE-323 coated capillary. Buffer used was ammonium acetate.

Compared to bare fused silica, EOF in PolyE-323 coated capillaries is less pH dependent and remains constant between pH 4-8, which provides reproducible migration behaviour and enables optimisation of pH for the separation without altering the EOF. PolyE-323 capillaries can be prepared with high reproducibility in coating performance (less than 4% RSD in EOF (n=10)).

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5 Coupling CE to mass spectrometry (CE-MS)

Mass spectrometry has today a central role in life science, as it has become the analytical technique of choice in many application areas. MS methods often provide high sensitivities of analysis and produces data that is characteristic for the structure of the analytes. The analysis of a sample with MS requires that the molecules are electrically charged (ionised) and in the gas phase. Heating of a sample to get it into the gas phase can easily be accomplished. However, analytes such as peptides and proteins are decomposed by heating. Therefore a "soft" ionisation/desorption technique is required to produce intact molecular ions in gas phase. With the introduction of soft ionisation techniques, such as ESI and MALDI, in the early 1980´s, MS has been applicable in the analysis of many biologically active compounds. Today, there are a wide variety of MS-based strategies suitable for the analysis of different types of samples. In protein and peptide analysis, the most commonly used techniques are MALDI-MS and ESI-MS. Although mass spectrometry has a good capacity to analyse complex biological samples, great improvements in sensitivity and selectivity can be achieved by implementing a separation step prior to MS analysis11. Furthermore, when ions are formed in the ion source there is always a competition for charge among the analyte molecules. Analytes with low affinity for charge and/or low abundance will be outclassed in the ionisation process, which results in signal suppression and a biased analysis. A separation step reduces the number of components that are introduced into the ion source simultaneously, thereby reducing signal suppression13,79-81. Capillary electrophoresis is a high resolving separation technique suitable for the analysis of small sample volumes and a wide variety of analytes. The combination of the two techniques that was first introduced about 20 years ago14 enables a two-dimensional separation, in electrophoretic mobility and size-to-charge ratio, in the same analytical system, providing a great potential for analysis of complex biological samples. The hyphenation of CE to ESI-MS and MALDI-MS will be explained more in detail below.

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5. 1 CE-ESI-MS Capillary electrophoretic separations of samples with biomedical origin are normally performed in liquid phase. ESI is an atmospheric pressure ionisation technique that transforms ions in solution into gas phase prior to mass spectrometric detection, as illustrated in Figure 7. ESI therefore ideally suits on-line coupling of CE to MS.

Figure 7. Electrospray ionisation in positive ion mode where the ions from liquid phase are generated/transferred into gas phase.

One of the biggest benefits of using ESI in combination with protein separation and mass spectrometry detection is that the analytes become multiply charged during the ionisation process. This effect is especially pronounced when acidic buffers (pH<pI) are used in the separation of proteins that are polyelectrolytes whose charge state is strongly dependent on pH. The multiple charge effect results in the decreased “weight” of proteins since it is expressed in mass over charge ratio (m/z). This is essential in the analysis of large proteins as many mass analysers have a limited m/z range, as demonstrated for 66 kDa human serum albumin in Paper II. Another factor of great importance when interfacing liquid separations to MS via ESI is also the speed of evaporation of the eluting liquid. The low flow rates used in CE facilitate a faster evaporation prior to introduction of analytes into the mass spectrometer. Furthermore, CE separation is based on ionisable analytes, which is highly compatible with the ESI process. A less straightforward situation can, though, come up when the requirements regarding the buffer compatibility have to be satisfied both for CE and ESI as typical CE buffers have a high ionic strength and low volatility while ESI requirements are the opposite. The coupling is therefore

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many times a compromise between high separation efficiency and optimal ESI conditions. Such limitations were circumvented in Paper II by using PolyE-323 modified capillaries. The separation of proteins in these capillaries could be rapidly performed resulting in high efficiencies using ESI compatible volatile background electrolytes. Due to the electrical contact, that in CE must be maintained at both ends of the capillary to define the electric field for the separation, there have been different approaches of coupling CE to ESI-MS. This thesis involves two of them, sheath-flow and sheathless interfaces14,82. These interfaces are shown schematically in Figure 8.

Figure 8. CE-ESI-MS interface designs: (A) sheath flow, (B) sheathless.

In a sheath-flow interface, a sheath-liquid is delivered at a constant rate co-axially to the CE effluent to provide electric contact at the capillary tip and to serve as an electrode. The sheath-flow interface allows an independent optimisation of the BGE and ESI solution and the sheath-liquid may have an important role in promoting the ionisation process in the electrospray. The ability to change the liquid composition post-separation can be very beneficial especially when a BGE with denaturing properties, such as acidic pH and/or content of organic modifier, can not be used during the separation. Acidification and addition of the organic solvents, to help the protonisation and the electrospray formation respectively, can thus be added post-column. This type of interface also helps a stable electrospray formation when electroosmotic flow rates are low and is necessary in applications where the EOF is completely eliminated. However, the addition of sheath-liquid in combination with high voltage may sometimes lead to changes in the EOF and cause so called moving ionic boundaries or changes in the pH due to electrode reactions83,84. Another consequence of adding extra flow is the dilution effect on the CE effluent and thereby decreased sensitivity85,86. These drawbacks are, however, often disregarded due to advantages of the

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sheath-flow interface that is considered stable and robust and is the most widely used interfacing technique today31,87. In the sheathless interface, the electric contact at the outlet end of the capillary is often defined by an outer conductive coating. In this type of interface, the outlet buffer vial can simply be replaced by inserting the capillary tip directly into the ion source of the mass spectrometer. The sheathless interface that was used in Paper II was achieved by depositing a coating of graphite containing polymer on a previously tapered capillary end tip. These ESI tips have been shown to be very robust88. Combination of such a stable conductive coating with a high EOF in this type of interface enables high sensitivities to be achieved as the dilution of the CE effluent is eliminated. When connecting CE separations of high efficiency with ESI-MS the speed of the signal acquisition needs to be high in order to maintain a good peak shape47. A time-of-flight (TOF-MS) instrument that fulfils this demand and enables detection over a wide m/z range has thus been chosen as detector in Paper II.

5. 2 CE-MALDI-MS Unlike ESI, MALDI is an ionisation technique that usually operates under high-vacuum conditions (<10-6 torr). In MALDI-MS the sample is deposited on a sample plate where it is mixed with or covered by a matrix of highly light-absorbing low-mass molecules. The ionisation of analytes is performed with the aid of laser pulses, of typically nanosecond duration. The matrix molecules are resonantly excited by the laser pulse and the absorbed energy causes translational motion and ionisation of analyte molecules in the gas phase. The principle of this ionisation technique is shown in Figure 9.

Figure 9. Matrix-assisted laser desorption/ionisation process.

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In contrast to ESI, MALDI creates mostly singly charged ions, which makes the spectral interpretation easier. The production of mostly singly and some doubly charged ions is a remarkable advantage in the analysis of complex samples, such as peptide fragment mixtures from protein digests. Although it is possible to analyse complex samples with MALDI directly, it is often the case that mixtures are too complex for unambiguous identification without a previous separation. The coupling of the CE separation that is compatible with essentially all buffers and analytes, to MALDI-MS is becoming an attractive approach in the analysis of complex biological mixtures. Although MALDI-MS is known to be more tolerant towards non-volatile electrolytes, in comparison to ESI-MS, their presence during the laser desorption/ionisation process can be strongly signal suppressive89. A way to address such problems is to use modified CE capillaries, as shown in Paper II-VI, which allows replacing of the CE typical non-volatile buffers with more volatile MS compatible ones without compromising speed and quality of the CE separation. Among all the different approaches that have been used in coupling liquid-based separations to MALDI-MS detection, the off-line methods have gained most popularity90-93. This is due to the difficulties to transfer solutions from the liquid-based separations on-line into the high-vacuum ion source of the MALDI. However, for the cases where it is preferable to keep the on-line connection of a liquid separation to MALDI-MS it has been possible to overcome this difficulty94,95. Off-line fraction collection is the simplest method of interfacing the separation in CE with MALDI-MS detection. It involves a subsequent but separate MALDI analysis of the sample fractions collected from CE. This approach allows the CE separation and MALDI-MS to work as two independent systems enabling individual optimisations of each of them. Another attractive feature of the off-line interfacing of CE to MALDI-MS is that the demand on the data acquisition in MS is not dictated by the fast separation in CE. This is of particular benefit for further analysis by MS/MS, which can be performed subsequently on the selected spots containing interesting compounds. In Paper III-VI an off-line automated micro-fractionation device was used with an incorporated sheath-flow arrangement, as shown in Figure 10.

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Figure 10. Off-line coupling of CE to MALDI-MS.

This interface allows a flexible and controlled fraction collection. Narrow zones of proteins from CE can be eluted in separated fractions thereby preventing the signal suppression79-81 and further increasing the spectral simplicity in MALDI-MS. On-target enzymatic digestion of selected protein fractions can be performed after MALDI-MS analysis, as demonstrated in Paper III. The rapid protein separation in CE and direct deposition of fractions on a MALDI target plate using off-line interfacing is a useful platform for rapid protein identification. The simple and robust approach significantly reduces the sample handling and potential loss compared to traditional tryptic digestion procedures. Furthermore, this approach opens possibilities of performing studies of intact protein isoforms and posttranslational modifications (PTMs), such as phosphorylation, glycosylation, acetylation, methylation and other modifications that may affect the function of proteins and play a significant role in diseases pathways96,97.

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6 Sample preparation

Common analytical procedures comprise several important steps: sampling, sample preparation, separation/isolation, identification and structural characterization, quantification, statistical evaluation and conclusion. Sample preparation is necessary to isolate the desired components from the sample matrix since most of the analytical methods can not directly handle the matrix. Moreover, introduction of matrix components into the analytical system can also cover up and hide the analyte signal of interest, which makes the analysis difficult or even impossible. Sample preparation can also include "clean-up" procedures for complex samples. In this step, also known as pre-concentration or enrichment, the analytes are concentrated to a suitable level at which they can be measured by the method chosen for the analysis. The choice of sample preparation method is crucial in chemical analysis since it is often the most critical and time-consuming step in the analytical chain. The isolation of proteins and peptides from biological matrices can be performed using various traditional sample preparation techniques such as homogenisation, centrifugation, precipitation, solvent extraction, liquid-liquid extraction, solid phase extraction, micro-dialysis and ultracentrifugation98. The evolving field of proteomics has created a need for development of new sample preparation methods99 since it is often important to perform a complete characterization of the natural distribution of analytes in a sample or a set of samples e.g. in clinical proteomics. Global identification of proteins in comparative proteomics studies, where the protein expression of a certain biological system is compared with another or the same system under perturbed conditions, is extremely complex. The interplay between sample components in different physiological processes and their relative abundances related to these processes do not allow for sample preparations where there is risk for sample loss if such differences in the sample composition have to be followed, as exemplified in Paper VI. Common difficulties that also can arise in clinical analysis are limited availability of biological material often in combination with very low concentrations of the molecules that are of interest to analyse. For such situations more straightforward protocols in sample preparation are desirable to avoid excessive purification steps, since loss of the material at each step is inevitable. Using one sample preparation technique is, however, most often inadequate for detection of proteins and peptides present at medium and low concentration levels in the sample. Combination of several sample

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preparation steps is thus normally necessary. Sample preparation approaches that involve fractionation, rather than purification, of the sample components based on e. g. pI or abundance to reduce the sample complexity, as in Paper IV and V, are thus to prefer in protein profiling of proteomic samples. Each sample fraction can thereafter be analysed separately which increases the possibility to resolve and visualise more of the entire protein content in the sample with minimised risk for sample loss. Irrespective of what the need is, to analyse specific proteins or to map the protein content, there is a lack of methods and instruments capable of identifying and quantifying components from complex samples in a simple, single-step operation. Individual components for separation, identification and quantification of proteins as well as tools for integrating and analysing all the data have to be used in combination, as illustrated in Figure 11.

Figure 11. Summarising flow chart of technologies used at each step in proteomic analysis of proteins and peptides. Abbreviations: LC, liquid chromatography; AC, Affinity chromatography; 2-D LC, two-dimensional liquid chromatography; 2-DGE, two-dimensional gel electrophoresis; CE, Capillary electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry; LC-ESI or MALDI MS/MS, liquid chromatography-electrospray ionisation or matrix-assisted laser desorption/ionisation tandem mass spectrometry; CE-ESI or MALDI MS/MS, Capillary electrophoresis-electrospray ionisation or matrix-assisted laser desorption/ionisation tandem mass spectrometry; LC-FTICR-MS, liquid chromatography–Fourier transform-ion cyclotron resonance mass spectrometry; UV, ultraviolet detection; Stable isotope labelling (see section 6. 2 .1).

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6. 1 Sample fractionation methods 6. 1. 1 Immunoaffinity depletion of high-abundant proteins A great challenge for the existing analytical techniques in the analysis of proteins and peptides in human body fluids has been the huge dynamic range of concentrations that these molecules occur in. The predominance of several high-abundant proteins, like albumin and immunoglobulins, has hampered the proteome analysis in such samples. For instance, a run of a tryptic digest from an unprocessed body fluid in a CE-MS experiment will result in peptides from high-abundant proteins distributed all over the electropherogram inducing suppression effects in the MS measurements. Furthermore, as the high-abundant proteins are dominating the sample, they limit the loading of other proteins in a proportional manner. Thus, the high-abundant proteins complicate the analysis and prevent the identification of proteins that are less abundant. Such difficulties have increased the need for the development of strategies for reducing the sample complexity to enable analysis of proteins and peptides present at much lower concentrations. This is due to that many known disease biomarkers have been found among the very low-abundant proteins100,101, which makes lower concentration ranges interesting for protein profiling. Simultaneous immunoaffinity depletion of multiple high-abundant proteins has been an attractive approach among the various strategies for reduction of sample complexity102-105. The most advantageous feature of such sample preparations is that they enable specific and selective removal of several of the most abundant proteins which together constitute up to 95% of the total protein content in biological fluids. A common concern in the depletion contexts has been the unintentional removal of low-abundant proteins along with the high-abundant ones due to protein-protein interactions. However, the depletion of high-abundant proteins by modern immunoaffinity methods has been reported as highly reproducible106,107. It is also important to emphasise that the removal of high-abundant proteins represents a subtraction of these proteins from the original sample. This allows for a separate analysis of the bound fractions that may contain non-targeted low-abundant proteins. In Paper IV, immunoaffinity depletion was applied to reduce the complexity of human cerebrospinal fluid (CSF) by depleting twelve of the high-abundant plasma/CSF proteins. The IgY-12 depletion columns used in our study were developed for depletion of high-abundant proteins in human blood plasma. According to our results, the depletion method is compatible also with CSF. Using IgY-12 depletion columns, the possibility to detect proteins at lower expression levels with CE and MALDI-MS/MS could be significantly improved. Of the 84 proteins identified in both CSF samples (depleted and non-depleted), 22 proteins could be identified with much higher scores and 34 proteins could be found only in the depleted sample.

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6. 1. 2 In solution isoelectric focusing The principle of isoelectric focusing (IEF) is based on the separation of molecular ions in a pH gradient formed in an electric field. The separation is limited to molecules that can be either negatively or positively charged (amphoteric), like proteins and peptides. At a certain pH value each molecule exhibits a net zero charge, which is referred to as isoelectric point (pI) and is characteristic for the molecule. Along the applied electric field a pH gradient is established and the molecules of a certain overall charge move towards the electrode of opposite charge until they reach the pH equal to their pI values20. The most common use of IEF is as the first dimension separation in two-dimensional gel electrophoresis (2DGE). Although IEF has also been performed in solution earlier the drawback of this technique has been the rather large scaled instrumentation requiring large sample volumes108,109. However, a new IEF instrument, called MicroRotofor, was recently introduced, that allows miniaturised in solution phase IEF (sIEF). A two-dimensional liquid phase separation approach is presented in Paper V for mapping of the protein content in human follicular fluid (hFF). The two dimensions involve sIEF in MicroRotofor as the first phase, and CE with PolyE-323 modified capillaries as the second phase of the separation. The MicroRotofor has a capacity of 2.5 mL of sample containing at least 0.2 mg/mL of proteins. The focusing can be performed on the samples with relatively limited availability and over a broad pH range (3-10) suitable for the analysis of highly complex samples. Depending on the amounts of sample loaded the sIEF can be completed within 2-3 h. In addition to the separation the preparative properties of the sIEF technique offer desalting of the sample in parallel. The approach in Paper V is described as an alternative to the traditionally used slab-gel-based strategies to reduce the overall sample complexity and elevate the proteins present at lower concentrations in the original sample. Hereby a truly comprehensive analysis of the entire protein content in the sample is enabled, including very hydrophobic, small and highly basic proteins. Another very important advantage of the presented approach is that it offers the possibility of two-dimensional in solution separation of intact proteins. Alternatively, the proteins contained in the selected pH fractions can be collected, enzymatically digested and analysed by MALDI-TOF MS to obtain a peptide map or sequencing information by tandem mass spectrometric analysis. The protein identification can then readily be obtained by database searching.

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6. 2 Chemical labelling 6. 2. 1 Multiplexed quantitative proteomic analysis with mass spectrometry using stable isotope labelling The proteome of an organism is dynamic and constantly changes in response to various environmental factors and other signals e.g. age and disease. This dynamics results in different patterns of changes in molecular levels. Consequently, not only the knowledge about the changes of a protein solely, but multiple proteins, is needed. To quantify such changes is the only way to assess the impact of a stimuli and the deviation from “normal”. The major method for targeted quantification that provides good sensitivity and possibility for verification and validation of a potential biomarker candidate on protein level has been enzyme-linked immunosorbent assay (ELISA)110. The difficulty with ELISA is that multiple protein biomarker candidates can not always be verified due to the lack of commercially available specific antibodies. Thus, MS-based quantitative proteome profiling that offers systematic and simultaneous investigation of large sets of proteins and peptides in complex samples has been introduced. Various strategies that can be applied on different levels (organism, cell, protein or peptide) have been developed. These strategies can be divided into two groups (1) relative MS quantification in vivo111 or in vitro112,113 and (2) absolute MS quantification114,115. The principle behind isotopic labelling approaches is to chemically modify the samples that are to be compared, before or after enzymatic digestion. For instance, one of the samples (light) is modified with a reagent containing naturally abundant stable isotopes and the other with an identical reagent in which several atoms have been exchanged with their stable heavy counterparts. The samples are then mixed, separated and analysed with MS. Due to that the light and the heavy reagents have very similar chemical properties, the differently labelled peptides will co-elute from the separation and generate a peak doublet in the mass spectrum differentiated by a mass shift between the isotopes introduced. In relative quantification approaches, the intensities of corresponding mass spectrometric signals are compared. This comparison provides a measure of the relative expression levels of the related peptides or proteins within the combined sample. The described labelling strategy, frequently called tagging, is possible to apply on multiple samples that can be analysed in parallel. One of the commonly used strategies for multiplexed quantitative proteomic analysis is iTRAQTM (isobaric tag for relative and absolute protein quantification)113, that allows up to 8 samples to be analysed simultaneously. The principle of iTRAQTM-based quantitative proteomic analysis is illustrated in Figure 12.

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Figure 12.(a) The components of the multiplexed isobaric tagging (iTRAQTM) molecule: a reporter group, a mass balance group and a peptide reactive group. The dashed lines indicate the cleavage sites in MS/MS. (b) The overall mass of the molecule is kept constant using differential isotopic enrichment. (c) Illustration of the four isobaric combinations (identical m/z in MS). In MS/MS the reporter ions appear as distinct masses (114-117 Da). The differences in their intensities represent the relative differences in the abundance of the peptide among the mixed samples.

Quantitative chemical analysis with CE has had a limited applicability due to the difficulties to control the sample injection. However, as the corresponding isotopically labelled sample components have identical chemical properties and the samples are labelled and mixed before analysis, the variations in the injection, separation and ionisation-MS analysis are minimised. This opens new possibilities for CE to be used in quantitative proteomic analysis. In addition, as it recently has been pointed out, differences in retention times of labelled peptides during chromatographic separation can occur116. With this in mind, CE, being an electrophoretic separation technique based on differences in charge-to-size ratio of analytes, gains new interest in quantitative MS-based proteomics. In Paper VI multiplexed quantitative proteomic analysis was, for the first time, applied using 4-plex-iTRAQTM labels in combination with CE and MALDI-TOF/TOF-MS. Ventricular cerebrospinal fluid (vCSF) samples collected at four occasions during the recovery of a patient suffering from traumatic brain injury (TBI) were analysed. The profiles resulted in 43 protein identities of which 6 showed changes in relative abundances that can be correlated to TBI processes as well as many other among the identified proteins could be related to the pathology of TBI.

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6. 2. 2 Lys Tag labelling for enhanced protein identification in MALDI-MS Competitive ionisation effects in combination with various parameters that determine the quality of the sample spots can greatly affect the performance of MALDI-MS, especially in the analysis of complex mixtures. In these contexts the chemical properties of the amino acids have been found to play an important role for a peptide’s signal intensity. It has been found that arginine-containing peptides ionise easier and result in much higher signal intensities in MALDI-MS compared to peptides that contain lysine117. The explanation to this difference has been the higher basicity of the arginine residue. For the analysis of the small amount of sample introduced in CE, that is further distributed in separate fractions on the target plate, the suppression effects during the ionisation process can be detrimental. An interesting solution to derivatise lysine-containing peptides was introduced by Peters et al.118, and was experienced as a useful tool in the characterization of lysine-containing peptides and de novo sequencing of unknown proteins with MALDI-MS119. The derivatisation is provided by tagging lysines with an imidazol compound, 2-methoxy-4,5-dihydro-1H-imidazole, also called Lys Tag. The compound reacts exclusively with the ε-amino groups of lysines (Figure 13) leaving the N-terminal amino group free.

Figure 13. Reaction of a lysine residue with 2-methoxy-4,5-dihydro-1-H-imidazole (Lys Tag)

The peptides containing labelled lysine(s) exhibit higher basicity and ionise even easier than the arginine-containing peptides. In Paper IV, Lys Tag was used to label tryptic peptides from the depleted CSF sample prior to CE MALDI-MS/MS analysis. In the derivatised samples additional proteins could be identified and the significance of identification could be increased for proteins which were previously identified without Lys Tag. Important to point out is also that the successful CE separations of the Lys Tag-labelled, highly basic peptides is a confirmation on the efficient and complete coverage of the inner capillary wall in fused silica capillaries with the PolyE-323 polymer.

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7 Proteomic analysis

Proteomics concerns the characterization of the full complement of proteins in a specific organism, tissue or cell type at a given time. In short, proteomics is a field that involves the identification, characterization and quantification of proteins. This challenge can be to analyse 10 000-30 000 protein variants expressed in a mammalian cell and characterise all of them at a given time. In an ideal situation the protein characterization would include cellular localisation and sequence analysis as well as identification of post-translational modifications and binding partners. This width of the proteomics field brings an enormous number of questions that need to be defined, addressed and answered. Hence, the choice of the representative samples as well as the methods for their analysis have been important issues to address. At present, there is no simple approach that can be used to study proteomic samples or to follow the ongoing bioactivities that can be related to disorders in the human body. Two dimensional gel electrophoresis (2-DGE), and liquid chromatography (LC), sometimes multidimensional LC, combined with MS can be discerned as two main approaches among proteomic methods for qualitative and quantitative sample control120,121. Despite tremendous contribution the 2-DGE method has made to the study of various proteomes it has a number of fundamental limitations. For instance, the range of proteins that can be separated in 2-DGE is limited excluding those with hydrophobic properties, high molecular masses as well as those with high/low pI values122,123. Moreover, the quantification by this technique is based on the assumption that one protein is present in each spot what is compromised by co-migration effects124. Using LC, on the other hand, generally implies long column-equilibration times, long total time of analysis and the need to use organic modifiers98. The most common LC approach for analysis of proteins is characterised by enzymatically digested complex protein mixtures and separation of the resulting peptides by one-, two- or three-dimensional chromatography followed by detection and identification of individual peptides using mass spectrometric peptide mass fingerprinting or tandem mass spectrometry (MS/MS). Despite the above outlined limitations and the considerable amount of labour involved together with rather large amount of sample required, these strategies are still dominating and are suggested to be, in principle, capable of detecting proteins at low concentration levels (down to low-femtomole level)125,126.

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Although a repertoire of useful proteomics technologies is currently available for different types of applications further technological innovations are beneficial in order to increase sensitivity, reduce sample requirements, increase throughput and thereby more effectively uncover various types of proteins.

7. 1 Analysis of proteomic samples with CE Capillary electrophoresis, able to provide fast and efficient separations of heterogeneous complex samples with low sample volume requirements, is becoming recognized as a suitable alternative among today’s bioanalytical approaches127. During the last years, the focus of research in the CE field has steadily been shifting towards the presentation of CE coupled to MS as a valuable tool for solving more and more sophisticated analytical questions. Among these, the fields of life science, such as proteomics and metabolomics, have been the most pronounced prospects of CE-MS128-131. Important to mention in this context are a few obstacles, which have been preventing CE from reaching a faster progress and a wider acceptance in life science up to now. The irreproducibility in analyte migrations caused by the adsorption phenomena in fused silica capillaries has probably been the most important one. A significant progress to control this problem has been made during the last decades including the solution presented in this thesis using PolyE-323 surface. The migration irreproducibility during the CE analysis of highly complex samples could be minimised, as shown in Paper V. The limited injection volume in CE in combination with the huge dynamic range of protein concentrations in biological samples has been an additional challenge. For this purpose a number of different methods have been introduced that can serve as suitable solutions in proteomic analysis with CE132-136. The new sample fractionation methods together with simple chemical modification for enhanced ionisation efficiency in MS, as presented in Paper IV, can be a useful approach to circumvent the drawback of low loadability in CE. The nature of the injection in CE, that can not offer high reproducibility, has also been an important topic, especially in quantitative chemical analysis. Although it is difficult to obtain the high reproducibility in CE, as observed in HPLC with fixed loop injection, several studies have shown that the consequences of this disadvantage are possible to minimise137,138. In proteomic analysis the dynamic nature of human proteome, as well as sources of technical variability, require repeated analysis of proteomic samples in order to draw any conclusions on the relation of the sample content to the analytical question139. With respect to that also the poor reproducibility of the CE injection in repeated analysis becomes negligible.

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The developments in the fields of proteomics and biotechnology have increased the need for suitable analytical techniques for the analysis of intact proteins. Despite the ability of CE to separate intact proteins a core issue in CE-MS analysis of proteins is whether the intact proteins should be analysed or not. In protein analysis there are two main tracks to follow (1) analysis of intact proteins or (2) enzymatic digestion of proteins and subsequent analysis of the resulting peptides with CE-MS. One of the advantages with the enzymatic digestion approach is that peptides are less complex molecules than proteins. Consequently, analyte-wall interaction becomes a less important problem. In addition, peptides also resist higher content of organic solvents without denaturation, which makes peptide analysis more straightforward in comparison to protein analysis. Working with peptide mixtures from protein digests is on the other hand associated with spectral complexity compared to when a sample containing intact proteins is analysed. However, a difficulty in the analysis of molecules within a wide range of molecular weights is that the software supplied with instruments and publicly available databases only allow a reliable evaluation of MS data that has been obtained on a previously digested sample. This is due to that enzymatic digestion of proteins improves the resolution and sensitivity of the mass measurements and also allows studying of very large proteins with conventional mass spectrometers140,141. Due to the complexity of proteomic samples the application of both approaches on the same sample is desirable i. e. when protein identification studies are followed by structural elucidation. The two approaches are demonstrated in Papers II and III by the analysis of both tryptically digested and intact proteins. In addition, using CE-MALDI-MS, as shown in Paper III, intact proteins could first be separated in CE at physiological pH, and fractionated onto the target where after on-target enzymatic digestion of the fractions could be performed followed by MALDI-MS and MS-MS analysis. When used in the described way CE has a great potential in studies where it is important to keep proteins in their natural state such as studies on protein-protein interactions, conformational changes and stability of proteins142-144.

7. 2 Analysis of human body fluids Body fluid samples contain a wide variety of analytes with different properties and huge dynamic range (~1010) of relative abundances. Besides the large number of proteins with different polarity, hydrophobicity and size over a range of several orders of magnitude, body samples also contain large quantities of other compounds, such as salts and surfactants145. This complexity presents significant analytical challenge. A difficulty that one also can face in working with such samples is that the sample amounts often are very limited. The accessibility to some human body fluids can also be

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limited to a short period of time or a certain situation, as is the case with human follicular fluid analysed in Paper V and ventricular cerebrospinal fluid in Paper VI, respectively. Such dual challenges of large number of analytes with tremendous differences in physical properties and limited sample volumes put high demands on the choice of the method for their analysis. To enable analysis of such complex biological samples a separation technique of highest efficiency and resolution and broadest application versatility is required. Highest resolutions can today be obtained either by employing multidimensional liquid-chromatography based separation techniques, that are also most widely used, or in one step using capillary electrophoresis126,145-147. Anyhow, the most promising approaches in the analysis of complex proteomic samples seem to be those where the combination of the complementarities of the electrophoretic and chromatographic methods are utilized148-150.

7. 2. 1 Cerebrospinal fluid Human cerebrospinal fluid (CSF) is mainly secreted in the choroid plexus, located centrally in the brain. CSF circulates within the ventricles and surrounds the brain and the spinal cord, as illustrated in Figure 14. In adults it is produced at a rate of 500 mL per day of which approximately 125 mL is constantly present in the central nervous system (CNS)151. CSF acts as a mechanical protection of the brain and as a medium for transportation of metabolically active substances and products from various tissues within the CNS. Due to the contact of CSF with the blood, via blood-brain and blood-CSF barriers and their active, but selective transport systems, the composition of proteins and metabolites in CSF differs from that of plasma. The total protein concentration in CSF (0.2-0.5 g/L) is approximately 100 times lower than in plasma, and constitutes to about 20% of CNS-produced proteins140,152. The study of the proteome and the biochemical composition of CSF is thus expected to give unique insights into the physiology of the brain and CNS upon disorders. The variation in concentrations of the CSF constituents, due to the rostrocaudal concentration gradient152,153, may consequently result in differences of the sample composition depending on the collection site (lumbar or ventricular). In order to avoid differences caused by gradients, the CSF collected from the same region should be pooled. The sampling of CSF, be it by lumbar puncture or ventricular drainage, is considered as invasive. The amounts of sample collected for routine diagnostic purposes are typically 3-5 mL140. For these reasons, CE is a good candidate among the analytical methods for the analysis of CSF, as well as it has been in other applications in neuroscience associated with invasive sampling and small volume analysis2,154. CSF was analysed by CE in Paper II, IV and VI to investigate the potential of using CE with PolyE-323 modified capillaries for complex proteomics applications.

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Figure 14. An illustration of the human central nervous system (CNS). The blue space and arrows represent the circulation of the cerebrospinal fluid (CSF) in the CNS. Collection of CSF via (a) lumbar puncture and (b) ventricular drainage (vCSF).

Figure 15. Human follicular fluid (hFF) formation during the follicular development.

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7. 2. 2 Human follicular fluid Human follicular fluid (hFF) constitutes the in vivo environment of the oocytea during its maturation and is produced during folliculogenesisb (Figure 15). Various reports have shown the importance of studying hFF in human reproductive processes. The chemical composition of hFF has been reported to reflect the stage of the oocyte development, the quality of the oocyte and the degree of its maturation153,155. In these processes proteins play an important role. Although new insights into the range of proteins expressed in hFF have been provided during the last years156,157, many connections between the multiple processes occurring during follicular growth are still not clear. There is thus a need for further examination of hFF, as well as other reproductive fluids, in order to understand the progressive changes occurring during the follicular development and pregnancy progression. The extended knowledge about these changes can be valuable in pregnancy-related research e. g. in vitro fertilization therapy (IVF)158 and studies of disorders in reproductive processes155. The composition of the hFF resembles that of blood plasma, in that it contains a wide variety of substances and in total protein concentration that is approximately 70 mg/mL. In this context, a blood-follicular barrier that regulates the selective transport of proteins has an important role158. Analytical techniques that offer high resolution and sensitivity are thus needed for identification of proteins in such a complex sample. In Paper V, sIEF combined with CE off-line MALDI-TOF/TOF-MS was presented as a reliable, low-sample-consuming approach for profiling of the protein content in hFF. This approach was demonstrated as a suitable alternative in the analysis of samples provided in limited volumes using invasive sampling techniques as well as an alternative to the traditionally employed slab-gel-based methodology for profiling of highly complex biological samples.

a Immature stage of ovum b Follicular development

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8 Concluding remarks and future aspects

A good separation strategy before mass spectrometric detection is crucial in a study involving the understanding of complex mixtures such as biological samples in proteomics profiling. This is due to that the good separation also provides good conditions to increase the confidence of protein identifications and to improve the coverage of proteomes. In recent years, many new separation approaches have been applied successfully in proteome analyses and have provided great improvements in protein identification, although complete coverage of proteins in complex samples has not been achieved. The development of new methods is thus important in order to meet the analytical challenges represented by the huge dynamic range of concentrations and the complexity in biological samples.

In CE, a problem that has hampered a reproducible separation of proteins and peptides has been the protein-wall adsorption phenomenon, especially emphasised with increased basicity of these molecules. An important strive in the development of new CE-MS methods for protein analysis has thus been the development of stable surface modifications for the CE capillaries. This thesis describes a contribution to this field by developing and employing a novel polyamine coating, PolyE-323, for improved separation performance of proteins in CE when coupled on-line and off-line to MS. With a simple coating procedure a robust surface is provided that enables fast and improved protein separation in comparison to bare fused silica capillaries. Using PolyE-323 modified capillaries a reproducible analysis of complex samples such as human body fluids is also enabled.

However, in addition to sometimes stressed drawback of the limited injection volume, CE, as other analytical techniques, faces the global proteomic problems of dynamic range and sample complexity. In the lack of tools to multiply proteins and peptides present at low concentration levels in a biological sample, comparable to polymerase chain reaction (PCR) in genomics, it is necessary to find new approaches that provide possibilities to visualise low-abundant molecules in a complex biological sample. Such approaches may instead involve appropriate sample preparation strategies and multidimensional separations to reduce the sample complexity as well as methods to enhance the sensitivity of detection.

This thesis also demonstrates the role of the sample preparation as a key to improve the possibilities of using CE-MS in the proteomic analysis of highly complex human body fluids. Finally, the thesis describes application

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of CE in quantitative proteomic analysis with MS and how CE takes advantage of the properties of multiplexing strategies to circumvent the drawbacks of the injection and analyte migration time irreproducibilities.

What can be said about the future of CE-based proteomic analysis?

As multidimensional separation systems will continue to be necessary in the analysis of highly complex proteomic samples, the complementary features of CE hyphenated to CEC, LC and MS will be useful. Also, the increasing interest in analysis of intact protein isoforms and post-translation modifications will benefit from the ability of CE to handle intact proteins. Furthermore, technical advances are shore to continue. The challenges of proteomics drive a rapid development of new sample preparation strategies as well as the performance and sensitivity of the new MS instruments is constantly improved. In addition, the trend to miniaturise analytical systems and decrease the sample needs will proceed. Due to that the experiments in capillaries can be transferred to analogous runs in channels in micro fabricated structures, like microchips, by relatively straightforward procedures this will remain a relevant application area for CE. Recalling the demands on a modern separation technique described in the abstract of this thesis, CE seems to be a separation technique before its time. With the further developments and improvements outlined above one could conclude that: The future works for CE!

“It would be naive to think that the problems plaguing mankind today can be solved with means and methods which were applied or seemed to work in the past. . .”

Mikhail Gorbachev, (1988)

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9 Summary in Swedish

Ytmodifierade kapillärer för kapillärelektrofores kopplad till masspektrometri Metodutveckling och undersökning av kapillärelektroforesens potential som ett proteomikverktyg Viljan och intresset att bättre kunna förstå oss själva och vår omgivning är drivkraften i forskningen. Vi vill kunna reglera och kontrollera de funktioner och processer som styr livet. För att kunna utforska dessa processer är det viktigt att utveckla nya metoder för mätning av substanser i olika prover på ett snabbt och pålitligt sätt. Ett högaktuellt område inom denna forskning är proteomik. Proteomik innebär en omfattande studie som inkluderar identifiering, karakterisering och kvantifiering av alla proteiner uttryckta i en cell, en vävnad eller en organism vid en viss tidpunkt. Proteiner, tillsammans med peptider, ansvarar för en mängd olika funktioner i kroppen och därför är skillnader i deras kemiska egenskaper mycket stora. Samtidigt ställer dessa olikheter stora krav på de analytiska metoder som används för att studera dem.

Kapillärelektroforetisk separation av proteiner och peptider Kapillärelektrofores (CE) är en separationsteknik i vilken olika substanser i provet separeras efter sina storlek-till-laddning förhållanden. Därför är CE en attraktiv teknik för analys av molekyler som har ett brett intervall av just dessa egenskaper, så som proteiner och peptider är. En annan egenskap hos CE tekniken är den minimala åtgången av prov vid varje analystillfälle (nanolitervolymer). Detta gör CE attraktivt när provmängden är begränsad, som det ofta kan vara vid analys av humana kroppsvätskor. CE är dessutom en snabb, enkel och billig separationsteknik som ger hög effektivitet på separationssignalerna. Ett problem vid användning av CE för analys av proteiner och peptider har dock varit deras adsorption till den negativt laddade kiselkapillärväggen i CE kapillärerna (reversibelt eller irreversibelt). Adsorptionen är mer uttalad för de proteiner och peptider som är basiska (positivt laddade).

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I Artikel I, har en lösning för detta problem presenterats genom att använda en ny polymer, PolyE-323, för att modifiera den negativt laddade innerytan i kiselkapillärerna och på så sätt minska adsorptionseffekten. En mer omfattande utvärdering av PolyE-323 ytans stabilitet har presenterats i Artikel II där separation av en blandning av modellproteiner med specifika egenskaper har använts.

Masspektrometri Masspektrometri (MS) är idag en av de mest avancerade detektionsteknikerna. Med MS mäts molekylmassan av substanser och uttrycks som massa över laddning (m/z) i ett masspektrum. MS ger maximal informations-noggrannhet på kort tid och med minimal förbrukning av prov. Hög robusthet och reproducerbarhet är andra egenskaper hos denna detektionsteknik som också möjliggör automatiserad datainsamling och därmed en snabb genomströmning av analyser. När MS kombineras med mjuka joniseringstekniker som elektrosprayjonisering (ESI) och matris-assisterad laser desorption/jonisering (MALDI) möjliggörs samtidig analys av proteiner och peptider med hög upplösning. En förutsättning för en känslig analys med MS är dock att provet separeras innan detektionen med MS. I Artikel II och III, presenteras separation av modellproteiner och peptider samt komplexa prover av human blodplasma och tårar med CE kopplad till MS via ESI respektive MALDI joniseringsteknik. I dessa studier visas möjligheterna av användning av PolyE-323 modifierade kapillärer i ett kombinerat system av CE-ESI-MS eller CE indirekt kopplat till MALDI-MS/MS inom proteinanalys.

Provupparbetning Humana kroppsvätskor är mycket komplexa prover som förutom proteiner innehåller salter, kolhydrater, lipider och olika kombinationer av dessa sammansatta i komplexa molekyler. Förutom komplexiteten förekommer molekylerna i provet i mycket olika koncentrationer som dessutom ändras konstant som resultat av påverkan från omgivningen, vid olika stimuleringar eller med tiden t.ex. ålder eller sjukdom. Det dynamiska området av proteinkoncentrationer i ett biologiskt prov är mer än 1010, vilket motsvarar en molekyl av ett visst protein och tio miljarder molekyler av ett annat protein samtidigt i provet. Med en av dagens mest avancerade detektionstekniker, MS, går det inte att identifiera så låga halter av ett ämne i ett biologiskt prov utan provupparbetning innan separation och MS detektion. Provkomplexiteten och det breda koncentrationsintervallet är därför två stora utmaningar inom proteomik. Dessa utmaningar har resulterat i en snabb utveckling av nya provupparbetningsmetoder. Beroende på det

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analytiska syftet skiljer sig dessa metoder åt både kemiskt och metodologiskt, men grundprincipen för många av dem är fraktionering av provet.

I Artikel IV, presenteras en proteomikstudie av proteiner vid låga halter i human cerebrospinalvätska (CSF), efter användning av en antikroppsbaserad provupparbetningsmetod innan CE-MALDI-MS/MS analys. Identifieringen av dessa proteiner har även förbättrats med hjälp av en derivatiseringsmetod (Lys Tag) för att öka känsligheten för proteindetektion med MALDI-MS/MS. På detta sätt visas att det går att använda CE i kombination med MALDI-MS/MS för analys av komplexa biologiska prover, trots den begränsade mängden prov som går att injicera i CE (vid dessa analyser ~140 nL upparbetat provmaterial per analys).

Parallell kvantifiering av proteiner med MS Ett område som har fått mycket uppmärksamhet inom proteomik idag är global kvantifiering av proteiner i flera biologiska prover parallellt (multiplexing). Metodiken bygger på inmärkning av proteiner/peptider med stabila isotoper. De senaste inmärkningsstrategierna möjliggör identifiering och kvantifiering av proteiner i 8 prover parallellt. Varje prov märks in med en molekyl som har samma strukturformel, men olika isotopfördelning i de olika proverna. Eftersom MS mäter skillnad i massa synliggörs koncentrationsskillnaderna av ett och samma protein i de olika proverna, efter bortklyvningen av den inbundna isotopmolekylen under MS/MS analysen.

I Artikel VI, presenteras en 4-parallell analys av humant ventrikulärt CSF (vCSF) från en patient med traumatisk hjärnskada (TBI). vCSF var insamlat vid 4 tillfällen under patientens tidiga återhämtningsperiod efter skadan. Prov från varje tillfälle märktes in parallellt före analys. I CE-MALDI-MS/MS analysen identifierades 43 proteiner varav flera visar på signifikanta kvantitativa skillnader i provet mellan de olika provtagningstillfällena. Dessa resultat korrelerar väl med tidigare studier som visat att dessa proteiner är väl förknippade med post-traumatiska processer vid TBI. Studien visar också att problem med reproducerbarheten vid injektionen i CE kunde kringgås med hjälp av denna form av parallellanalys av proverna.

Resultaten som presenteras i denna avhandling visar tydligt på potentialen

av CE i kombination med MS som ett användbart proteomikverktyg för analys av komplexa biologiska prover.

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10 Acknowledgements

Finally, I would like to thank all the people who, in one or another way, have helped me through the years of this work. Special thanks to:

My supervisor, Prof. Jonas Bergquist, for financing my play in the lab for 5 years, and for your commitment and trust during these years. It has been a real pleasure to be a part of your group.

Prof. Karin Markides, for your enthusiastic and inspiring personality and because you supported me in the idea to start my PhD studies.

My co-supervisor, MagnusWetterhall, for your every-day-good-mood attitude and, of course, your commitment and help during the last years of this journey.

Jonas and Monica Waldebäck, who, according to the chronological order should be first on this list. For your help and support to find the best way to good analytical skills (should I have them now?). Ulf Hellman, Eva Heldin and Lennart Söderberg, for nice collaborations. Roland Petterson, for having answers to all mysteries of chemistry, and for loan of all the worlds’ cables and tools that one, as a chemist or non-chemist, might need. (It wasn’t that stupid to have the office next to yours). All my friends and relatives, past and present colleagues and staff at the section of Analytical Chemistry for five unforgettable years. Even though it is difficult to distinguish few from other colleagues I have to give special thanks to Sara Ullsten, My Moberg, Jörg Hanrieder, Titti Ekegren and Emilia Hardenborg, for sharing experiences both within and outside the world of Analytical Chemistry.

Sushi-companions, for sharing these moments as the highlight of the day.

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Foundations of Knut and Alice Wallenberg, C. F. Liljewalch and Anna Whitlock, and to Swedish Chemical Society, for granting the travel stipends that made it possible for me to participate in international conferences and broaden my research view throughout the time of my PhD studies.

The staff at Ulleråkersförskola, for taking care of my son during the days in such a good way.

Linda and Helene, for a good time and nice experiences during the studies.

Alma and Senad, for your friendship and the true curiosity about my research, for looking forward to the end of this work and a dissertation party in Uppsala. Will be! Mina föräldrar, för att ni alltid haft tilltro och respekt för mina val.

My son Ishak, for all the happiness you bring into my life.

And last, but not least, my beloved Suvad, for sharing worries and happiness with me for almost half of my life.

Uppsala, January 29th, 2009

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