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
Tutorial Day- 20100908 // René Brunisholz• • •
>>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
.2
* Salmonella plate 28012010\0_E14\1\1SLin
8
4x10
Inte
ns. [
a.u.
]
4182
.9
8469
.6
2
4
6
MALDI-MS apparatus
8518
.6
4818
.94296
.1
8640
.2
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
.8
9435
.0
08
1.0x
0
1.x104
5685
.1
4517
.7
6546
.8
3143
.928
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90.3
4.2 .8
0.4
0.6
0.8
0.
0.
0.
Inte
ns.
.
.
2
379
3464
7581
6929
.6
2258
.7
6082
.1
9145
.7
4999
.6
2526
.6
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.2
5308
.0
4082
.9
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258.
7
6082
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7
4999
6
2526
.6
7046
5308
.0
4082
9
6770
.2
0.
0.
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
Tutorial Day- 20100908 // René Brunisholz• • •
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)
5382
.0
4364
.5
55.86000
6138
.7
6719
.2
1.5
Sa o e a e e ca ( ) ( )62
5
6353
.8
4000
tens
. [a.
u.]
6001
.7
3375
.7
1.0
ens.
[a.u
.]
9522
.6
4761
.9
7159
.1
2000
Int
7763
.1
9069
.2
7371
.4
3499
.30.5
Inte
7261
.6
7717
.9
3579
.5
8369
.1
5614
.3
4184
.5
9239
.2
5143
.139
8993
.1
1028
4.9
3859
.0
1006
6.1
0
2994
.4
9964
.3
3889
.2
8471
.7
4542
.3
5269
.0
1184
3.7
1051
9.8
4990
.7
4106
.3
8101
.5
0.0
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
2.3. Biotyper ™ Differentiation between three L. brevis strains
Illustration of the MALDI results in the gel band view
Tutorial Day- 20100908 // René Brunisholz• • •
R i i h BiReporting with Biotyper
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
Biotyping: FGCZ Service ?
• MALDI-Biotyping
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
PAC Spotting of a Phosphorylated Peptide (2282.02 m/z)Storage Time at RT > 1 month >> MSMS
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
MALDI-MS MALDI MSMSMALDI-MSMS
Workflow: Label Localization Validation by MSMS
MSMS of 450 nm absorbing Peptideg p
Tutorial Day- 20100908 // René Brunisholz• • •
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
Tutorial Day- 20100908 // René Brunisholz• • •
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