PtiA l iG Protein Analysis Group - fgcz-intranet.uzh.ch

P t i A l i G

Biotyping and LCBiotyping and LC--MALDI specific ApplicationsMALDI specific ApplicationsProtein Analysis Group

Tutorial Day @ FGCZ 20100908y @

René BrunisholzlProteinAnalysisGroup PAG

Functional Genomics Center ZürichFGCZ ETHZ UNIZH

email: [email protected]

Identification Identification and classification and classification of of microbesmicrobes........................

I. Whole Cell Biotyping by MALDI-TOF (BiotyperTM) microbesmicrobes........................

(in collaboration with Agroscope, Wädenswil,PI : Dr. David Drissner)

??- AimFast and efficient identification of undesired and human pathogenic microorganisms in food

•

?? products of plant originOne Focus is related to Salmonella Typing…

- Agroscope-FGCZ Project:Establish fast and efficient workflow for the identification of microorganism based on full cell

t i t iprotein typing

Tutorial Day- 20100908 // René Brunisholz• • •

M th d f th Id tifi ti d Cl ifi tiMethods for the Identification and Classification of Microbes............

-Morphological aspects.....

-Phenotypical aspects ....

-Ribotyping (rRNA)

-Immunological assays (Serotyping)g y ( yp g)

-Small molecule biomarker profiling

-PulsedFieldGelElectrophoreseis PFGEPulsedFieldGelElectrophoreseis PFGE>>>gold standard in studies of pathogenic organisms

Separation of large DNA fragments

-Protein profiling >>> BiotypingMALDI TOF whole cell protein screening


>>based on the detection of mainly ribosomal proteins>>constantly expressed as high-abundant proteins.

1 P i i l f h l P t i MALDI MS1. Principle of whole Protein MALDI MS

Protein Matrix

dissolve sample dissolve matrix

of matrixca. 100000 fold excessof matrix

pipetting Laserpipetting LaserLight

8370

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* Salmonella plate 28012010\0_E14\1\1SLin

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MALDI-MS apparatus

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.6

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.94296

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03000 4000 5000 6000 7000 8000 9000 10000 11000

m/z

2. Principle of whole cell MALDI MS

1. Sample preparation 2. MALDI-TOF analysis

4716

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9435

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3000 5000 7000 9000 11000m/z

Tutorial Day- 20100908 // René Brunisholz• • •3. Identification: with BiotyperTM

2.1. MALDI-Biotyping Procedure• bacterial cell material from single cell colony suspended in water

• precipitation with ethanol

• extraction with 70% formic acid /acetonitrile.

• extracts (1 µl) were spotted onto the MALDI target

• addition of matrix (CHCA)

• mass spectra acquired on a Bruker Autoflex II mass spectrometer

(positive ion mode, 2-20 kDa)

d t l t d i th B k MALDI Bi t SW ( l d t dB)• data were evaluated using the Bruker MALDI Biotyper SW (coupled to a dB)

>>> once the extract is ready it will

take less than 10 minutes to obtain an ID


2 2 MALDI TOF Spectra of2.2. MALDI-TOF Spectra of - Salmonella enterica (A) and - Penicillium oxalicum (B)- spectra in linear positive mode- spectral range: 3000 – 12000/14000 m/z 8000

x104

Salmonella enterica (A) Penicillium oxalicum (B)

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4000 6000 8000 10000

m/z

4000 6000 8000 10000 12000

m/z

2.3. Biotyper ™ Differentiation between three L. brevis strains

list of organism

Results window of the Biotyper software

st o o ga s

Score2.40

Strain A

Strain B

Strain C


2.3. Biotyper ™ Differentiation between three L. brevis strains

Illustration of the MALDI results in the gel band view


R i i h BiReporting with Biotyper


MALDI Biotyping

Method characteristics• Applicable for bacteria, yeast and

fungi

Possible Users• Medical Microbiology (e.g. clean

room monitoring)fungi• Easy and fast sample preparation• Quick and reliable results

room monitoring)• Diagnostics (e.g. urine)• Food industry

• Identification down to strain (sub-specis) level possible

• High sample throughput

• Biotechnology• Microbiolgy

g p g p• Low running costs• Library set-up (Biotyper, Bruker)

and modifications by user possible

• >> ....you ??

and modifications by user possible


Biotyping: FGCZ Service ?

• MALDI-Biotyping


17

II.Interplay cap-HPLC and MS /MSMS

Mass spectrometer:Bruker Ultraflex TOFTOF:

HPLC :Agilent 1100 with microspotter

T ki t t it i t dT ki t t it i t d R t i ti f MSR t i ti f MSTracking target sites in tagged Tracking target sites in tagged proteins with e.g. a proteins with e.g. a DiodeArrayDetectorDiodeArrayDetector

Restriction of MS Restriction of MS analysis on tagged analysis on tagged peptidespeptidesDiodeArrayDetectorDiodeArrayDetector

(full spectrum 200(full spectrum 200--900 nm)900 nm)peptidespeptides


Interplay capHPLC <-> MS /MSMS /Edman Sequencing : Different Types of Target Supports

Sample spotSample spot

CalibrantCalibrantCalibrantCalibrant

Steel MALDI plate PAC-MALDI plate (disposable)

PVDF sheet (homemade support

MS and MSMS:-intact proteins

MS and MSMS:-peptides

Edman sequencing:-intact proteinsintact proteins

-peptides-sugars

peptides intact proteins -peptides

-aoTutorial Day- 20100908 // René Brunisholz• • •

II. LC-MALDI Specific ApplicationsSuited for In-Depth Analyses of Proteins / Peptides with respect to:

PTM:-Alignement of cystine bridges in proteins

Ph h it l li ti ith 32P- Phosphosite localisation, e.g. with 32P- Cofactor ID/ localisation (heme, bilines ao)- Carbohydrates

-localization within a protein-structure identification/verification

Label identification /localization /quantification:- fluorescent label (e.g.FRET analyses)

- identification of free Cys in a proteinwith e.g IAEDANS agent


A l i f Ph h t i /P tid

Case study 1:

Analysis of Phosphoproteins /Peptides Which are the Key Issues ?

• Phosphoproteins can be present at low abundance• Low stoichiometry of phosphorylation • Protein may exist in differentially phosphorylated forms• In MS measurement phosphopeptides usually appear with a low S/N ratio.• >>>> Use of 32P labeled proteins• >>>> Use of 32P labeled proteins......

??????Which method

should be applied?

??????

Tutorial Day- 20100908 // René Brunisholz• • •Kinase substrate How to catch the phosphorylated

peptide(s)most efficiently ?

20Ph h it M i 32P L b l d Ki S b t t ith PAC

32P labeled

Phosphosite Mapping 32P Labeled Kinase Substrate with PAC:Working Principle

kinase substrate

SDS-PAGEin-gel digestion

„traditional“ approach

„ “ PAC “ approach

Reversed-phaseHPLC

Reversed-phasecapillary LC

approach pp

Separation of peptides

Automatic fraction collection&

liquid scintillation counting

Microfractionation onto pre-spotted Anchorchips (PAC).

Direct exposure to X-ray filmX-ray film

Analysis of target spots by MS and

MS/MS

Concentration of radioactive fractions

Manual mixing of sample with matrix

Manual spotting onto ...Sample loss...

p gMALDI target plate

Analysis of samples by MS and MS/MS

calibrant spot

sample spot

Detection of Phosphopeptide 963.59 Da and the Phosphosite (Ser485) in 1AMPK

- Tryptic digest

- about 1.2 pmol

of 32P-labeled phosphopeptide

- Exhibiting about 2-3 Bq / ul

Separation on CapLC Agilent 1100

flow rate: 7 ul/minF ti ti 2 33 l/ t

Separation on CapLC Agilent 1100

RPC C-18 , 0.5 mm ID x 150 mm L

Fractionation : 2.33 ul/spot

Microfractionation onto a MALDI PAC-Target Plate (Bruker)Tutorial Day- 20100908 // René Brunisholz• • •

30

Summary : Imaging Phosphopeptides on

Cap LC profile MS/MS of 963 592 Da of spot C9 (loss of 98 Da)

Summary : Imaging Phosphopeptides on Prespotted Anchor Chip MALDI Plates

Cap-LC profile MS/MS of 963.592 Da of spot C9 (loss of 98 Da)

PAC autoradiography-profile Mass spectrum of spot C9ASMS 2006, Seattle / R.Türk et al., Optimizing Isolation and Mass Spectrometric Analysis…...R.Türk et al., Anal. Biochemistry 2009


PAC Spotting of a Phosphorylated Peptide (2282.02 m/z)Storage Time at RT > 1 month >> MSMS


Request: Localization of two chemically different labels Case study 2:

q ywithin a 15 kDa protein

• A target protein has been labeled with 2 chemically different chromophore labels :One absorbing at 350 nm, the other at 450 nm

• The label are either at position Cys 9 or at Cys 79

• Setting up a possible workflow to proof their localization experimentally within the protein

• How to proceed ? Cys9 Cys79


Possible Workflow : Label Identification

Fragmentation of target protein e.g. by trypsin or pepsin

Separation of the tryptic fragments using RPC on C18 Separation of the tryptic fragments using RPC on C18

Monitoring the peptide elution at different wavelengths

DAD diode array detector : identify the chromophore

containing peptidescontaining peptides

Microfractionation directly onto MALDI target plate

MALDI MS analysis of tagged peptides

validation of found precursors by MSMSTutorial Day- 20100908 // René Brunisholz• • •

MALDI-MS

Workflow: Label LocalisationDAD diode array detector : identify the chromophore containing peptides

MALDI-MSMS

AB

Full spectrum of a

Full spectrum of a fraction with 450 nm absorption

Full spectrum of a fraction with 350 nm absorption


MALDI-MS MALDI MSMSMALDI-MSMS

Workflow: Label Localization Validation by MSMS

MSMS of 450 nm absorbing Peptideg p


C CConclusion: Covalent Protein-Prelabeling MethodsCombined with LC-MALDI Analyses

• By using covalent protein-prelabeling methods MS based analyses become restricted to the target sitesD ti d i l i ti (l t !)• Dramatic decrease in analysis time (less spectra…..!)

– Combining autoradiographical imaging of 32P labelled peptides on pre-spotted Anchorchip with MS and MSMS analyses yields a rapid and efficient workflow for phosphosite determination

– Peptides containing labeled amino acids (at Cys / Lys etc) can be directlyPeptides containing labeled amino acids (at Cys / Lys etc) can be directly visualized by e.g. a Diode ArrayDetector DAD with a full spectrum (e.g. 200-900 nm)

• Storage time of spotted samples on PAC MALDI plate : several months– Re-analysing samples possible


Protein Analysis GroupThanks to.....

YolandaYolanda AuchliAuchliYolanda Yolanda AuchliAuchliBirgit Birgit RothRoth

SergeSerge ChesnovChesnovDavid DrissnerAgroscope Wädenswil

Serge Serge ChesnovChesnovSimone WüthrichSimone Wüthrich

Peter HunzikerPeter HunzikerBritta Stoop

Frank Hesford

Roland TürkRoland TürkDietbert NeumannDietbert NeumannDietbert NeumannDietbert Neumann

Ramon ThaliRamon ThaliTheo WallimannTheo Wallimann

[email protected]

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