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Mass Spectrometry in an “Omics” World. ME.330.804
Lecture 10: Quantitative ProteomicsWednesday, November 14, 2012
Robert N. Cole, Ph.D.Mass Spectrometry and Proteomics Facility Johns Hopkins School of Medicine371 Miller Research Bldg (formally BRB)Ph: (410) 614-6968
email: rcole@jhmi.edu
Quantitative ProteomicsQuantifying Individual Proteins in Complex Mixtures
(Functional Proteomics)
Changes in protein levels(biomarkers, pathways, binding partners)
Applications
( p y g p )
Changes in protein modifications (structure/function)
Changes in subcellular localization (trafficking)
Kinetics (protein turnover, modification dynamics)
Quantitative Proteomics Methods
Label-Free Labeling
ChemicalMetabolic Enzymatic
DIGE (Difference Gel Electrophoresis)Radiolabeling 18O-Labeling
Relative Absolute
ICAT (Isotope-Coded Affinity Tags)
TMT (Tandem Mass Tags)
iTRAQ (Isobaric Tags for Relativeand Absolute Quantitation)
SILAC
DensitometryPeak IntensitySpectral Counting
AQUA (Absolute Quantification)MRM (Multiple Reaction Monitoring)QconCAT (Quantification Concatamers)PSAQ (Protein Standard Absolute Quantification)SWATH
Best Approach?
All approaches are: Complementary
different separation techniquesdifferent sets of proteins identified
There is NO one best approach.
Technically challengingsample preparationdata acquisitiondata analysis
Require fractionation to dig deeper into proteomelimited by dynamic range
2
Best Quantitative Proteomic Experiments
Defined question (i.e. hypothesis driven)
Defined system
Independently measurable phenotype
Sample preparationReproducibleScalableCompatible buffer system
Sample Preparation(Most Important Step!)
Reproducibility
Fractionation(reduce protein amount and complexity,enrich for target proteins)
Buffer Exchange(remove non-compatible buffer reagents)
Must Be Reproducible and Standardized!
Biological Replicates
1 2 3
InitialProtocol
StandardizedProtocol
1 2 3
Reduce Sources of Variability:Technical: “good” < “bad hands”
few < many steps
Biological: cells < tissue < body fluidsyeast < nematode < human
Fractionate to Detect Low Abundance Proteins
MWkD
115
MW
S
BS
A
180
Total Serum
Top 6 proteins removed
1158264
4937
261915
6
4-12% SDS-PAGE, SimplyBlue stain
3
1D
pI3.0 11.0
250150
75
50
37
25
10
pI3.0 11.0
250150
75
50
37
25
10
250150
75
50
37
25
10
2D
versus
7 cm versus 24 cm
10.53.5 5.8 8.2
Buffer Reagents CompatibilityDepends on the Proteomic Method
Three non-compatible categoriesDetergentsSaltsReagents that compete for label
Buffer exchange to make compatibleFiltrationPrecipitationBinding Affinity(Dilution)
Detecting low abundance proteins requires“Clean samples”, no interfering reagents
Quantitative Proteomics Methods
Label-Free Labeling
ChemicalMetabolic Enzymatic
DIGE (Difference Gel Electrophoresis)Radiolabeling 18O-Labeling
Relative Absolute
ICAT (Isotope-Coded Affinity Tags)
TMT (Tandem Mass Tags)
iTRAQ (Isobaric Tags for Relativeand Absolute Quantitation)
SILAC
DensitometryPeak IntensitySpectral Counting
AQUA (Absolute Quantification)MRM (Multiple Reaction Monitoring)QconCAT (Quantification Concatamers)PSAQ (Protein Standard Absolute Quantification)SWATH
Label-Free Methods
No additions to analysis
Separate analysis of Each sample
Biological Variability
Quantitative Proteomics Methods
Technical Variability (Sample Prep and MS analysis)
4
Bacterial Strain #1 Bacterial Strain #2
pIMW
Label-Free: Densitometry - 2D Gels
Compare spots from different gelsQuantify by relative spot volume (or number of peptides)Gel reproducibility and spot matching criticalGel warping for matching spots can warp out modificationOften more than one protein per spot
Label-Free: Spectral Counting -1D Gels
21
Sample
Slice
2 Sample 1 Sample 2
22 Polymereic Immunoglobulin Receptor 15 5
22 Transferrin 5 1022 Vanin 1 5 5
21 1B-glycoprotein21 Complement component 5
21hGC-1 (human G-CSF-stimulated clone-1
21 1 G F bi di t i
Gel Slice Number
Protein IdentifiedNumber of Spectra
Molecular Cell Proteomics 3:715, 2004.
Comparing protein bands on same gelQuantify by number of peptide spectra from protein At same MW, more spectra = more protein
21 1gG Fc-binding protein21 Mac-2-gining protein
18 antichymotrypsin18 Albumin
Label-Free: Peak AreaQuantify each protein from:
intensity of 3 most intense peptidesaveraged from 3 technical replicates
Each sample separate LCMS/MS experiment
m/z
3DPeak Intensity
Map
sity
Time
Annotate“Spots”
Reproducibility of LCMS/MS is critical
Time (min)
Inte
ns
Compares multiple LCMS/MS experiments
Label-Free: Spectral CountingQuantify each protein from:
number of peptides from protein number of spectra for each peptide
Each sample separate LCMS/MS experiment
sity
n N
um
be
r
Compares multiple LCMS/MS experiments
Reproducibility of LCMS/MS system critical
Time (min)
Inte
ns
Sample
1 32 4Protein Name Ac
ce
ss
ion
MW
5
Gels
Cells Tissue or Fluid
Label-Free Workflows
Protein Extract
ProteolysisGel matching
Fractionation
MSPeak Area
Spectral Counting
Confirmation
MS2
MS3
Peptide/Protein ID
g
Densitometry
Quantitative Proteomics Methods
Label-Free Labeling
ChemicalMetabolic Enzymatic
DIGE (Difference Gel Electrophoresis)Radiolabeling 18O-Labeling
Relative Absolute
ICAT (Isotope-Coded Affinity Tags)
TMT (Tandem Mass Tags)
iTRAQ (Isobaric Tags for Relativeand Absolute Quantitation)
SILAC
Gel matchingDensitometryPeak IntensitySpectral Counting
AQUA (Absolute Quantification)MRM (Multiple Reaction Monitoring)QconCAT (Quantification Concatamers)PSAQ (Protein Standard Absolute Quantification)SWATH
Non-Labeling Methods
No additions to analysis
Separate analysis of Each sample
Biological and Technical Variability
Quantitative Proteomics Methods
Labeling Methods
Typically adds steps to analysis
Simultaneous analysis of Many samples (Multiplexing)
Biological Variability, Reduces Technical Variability
Cuts instrument time (data collection) by 50-75%
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
Metabolic Labeling: No Technical Variability
12C-Arg 13C-Arg
Mix Cells
12C-Arg 13C-Arg
Mix Cells
12C-Arg 13C-Arg
Mix Cells
Control Treated
Only for cell cultureNo added steps
Ong et al. J P t RMix Cells
Isolate proteins and digest with trypsin
Peptide ID and quantify by tandem MS
Mix Cells
Isolate proteins and digest with trypsin
Mix Cells
Isolate proteins and digest with trypsin
Peptide ID and quantify by tandem MS
No added stepsStandard Deviations 5%
Quantify mass pairs
J Proteome Res 2: 173, 2003
6
Quantify by 13C to 12C-Arg-labeled peptide ratios
J Proteome Res 2: 173, 2003
By using13C and 15N Arg:Can increase mass difference to 9 Da,Analyze 2 to 4 samples per experiment
Chemical Labeling:Difference Gel Electrophoresis (DIGE)
Disease Protein ExtractLabel with fluor 2
Control Protein ExtractLabel with fluor 1
Separate proteins on one 2-D gel
Mix labeled proteins from both samples
p p g
Excitation wavelength 1
Excitation wavelength 2
Image gel
View differences
Unlu et al. Electrophoresis 18:2071, 1997
Protein comparison in a single gel!
Three Fluorescent Cy Dyes
Cy2, Cy3, Cy5
-amino group of lysine
Matched for Charge and MW
Third Cy Dye used as an Internal Standard
Cy2
Cy5
Cy3
Third Cy Dye used as an Internal Standard
All possible protein spots overlaid on every gel.
Simplifies gel to gel matching.
Each spot has it’s own internal standard spot fornormalizing across gels.
Reduces experimental variations.
Accounts for differences in sample load.
Separate proteins on one 2-D gel
Mix samples
DIGE Analysis Pooled Samples labeled with Cy2(on all gels to compare one gel to another)
Disease Protein ExtractLabel with Cy5
Control Protein ExtractLabel with Cy3
p p g
Excitation wavelength 1
Excitation wavelength 2
One additional stepReduced technical variabilityQuantify intact proteinsCells, tissue, fluids
7
1
2
5
6
9
10
13
14
3
4
7
8
11
12
15
16
Enzymatic Labeling: 18O-labeling ProtocolExperimental Protein Extract
Control Protein Extract
Trypsin Trypsin + 18O-water
Anal. Chem 73:2836, 2001J Proteome Res 2:147, 2003
Cells, tissue, fluidsNo added stepsNo MS technical variability
Variability in protein extractionStandard deviations 10-20%Loss of label to water
Mix labeled peptides
Peptide ID and quantify by tandem MS
C-terminal C-terminal(adds 4 Da)
Quantify by 18O to 16O peptide ratios
Peptide sequencePeak intensity ratio
± errorNWAAFR 2.5 ± 0.1GNVYWVR 3.4 ± 0.7GVIETVYLR 3.0 ± 0.3LLTPNEFEIK 3.1 ± 0.5VAITFDSSVSWPGNDR 2.9 ± 0.4NLLLLPGSYTYEWNFR 2.8 ± 0.2
(4%)(20%)(10%)(19%)(14%)
(7%)
Only 4 Da mass difference18O exchange with waterCompares only 2 samples per experimentNo good analysis software yet Anal. Chem 73:2836, 2001
Chemical Labeling: Mass Tags (in 3 Flavors)
Mass Tag CompanyNumber of Tags
4-plex iTRAQApplied
Biosystems4
8-plex iTRAQApplied
Biosystems8
TMT ThermoFisher6
iTRAQ = Isobaric Tag for Relative and Absolute Quantitation
TMT = Tandem Mass Tags
8
iTRAQ Tags(Isobaric Tag for Relative and Absolute Quantitation)
Peptide Reactive
Reporter Balance PRG
Reporter Mass Balance Mass
Isobaric Tag(Total mass = 305)
Eight possible tags
Group(binds to amines)
113114115116117118119121
192191190189188187186184 Ross et al. 2004 MCP 3:1154
Pierce et al 2007 MCP 7:853Charged Neutral
Label Peptides
Peptides:
8-plex iTRAQ Workflow
Proteins:
S2114
S3115
S4116
S5117
S6118
S7119
S8121
S1113
S2 S3 S4 S5 S6 S7 S8S1
S2 S3 S4 S5 S6 S7 S8S1
All MixedPeptides
Analyze each SCX Fraction by LC-MS/MS
Fractionate Peptide Mixture on SCX or bRP Column
Mix Labeled Peptides
114 115 116 117 118 119 121113 Mixed
272.17434.25
1173.58
Gates_ITRAQ_8Plex_SCX-1#6263 RT: 62.61 AV: 1 NL: 6.05E5T: FTMS + p NSI d Full ms2 1130.61@hcd45.00 [110.00-2000.00]
50
60
70
80
90
100
bu
nd
an
ce
Fragmentation SpectrumiTRAQ-EESPLLIGQQSTVSDVPR
Fragmentation Spectrum
y2272.17 b1
434.25 y111173.58
b3
Sequence b-ionb-ion mass
y-iony-ion mass
iTRAQ-E 1 434.255 18 2259.210E 2 563.298 17 1825.966S 3 650.330 16 1696.923P 4 747.383 15 1609.891L 5 860.467 14 1512.838L 6 973.551 13 1399.754I 7 1086.635 12 1286.670G 8 1143.656 11 1173.586Q 9 1271.715 10 1116.564Q 10 1399.773 9 988.506S 11 1486.806 8 860.447T 12 1587.853 7 773.415V 13 1686.922 6 672.368S 14 1773.954 5 573.299D 15 1888.981 4 486.267V 16 1988.049 3 371.240P 17 2085.102 2 272.172R 18 2241 194 1 175 119
Predicted Fragmentation Masses
650.33563.29
860.46 1609.89
175.12 973.551286.67
1955.011696.921399.75
372.24 1826.97
747.38 1086.64 1512.83
672.37988.50
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
Re
lati
ve A 650.33b2
563.29b5/y8860.46
y151609.89
y1175.12
b6973.55 y12
1286.67y18 – iTRAQ
1955.01y161696.92
b13/y131399.75
372.24y17
1826.97b4747.38
b71086.64
y141512.83
y6672.37 y9
988.50
R 18 2241.194 1 175.119
S3
S5
S6 S8
S1
Gates_ITRAQ_8Plex_SCX-1#6263 RT: 62.61 AV: 1 NL: 6.05E5T: FTMS + p NSI d Full ms2 1130.61@hcd45.00 [110.00-2000.00]
50
60
70
80
90
100
ve A
bu
nd
an
ce
117.11
115.11
113.11
Eight reporter ions quantify same peptide from eight samples in
one spectrum
70,000 Interpretable Spectra per iTRAQ Experiment(5% False Discovery Rate)
S2S4 S6
S7
S8
112 113 114 115 116 117 118 119 120 121 122m/z
0
10
20
30
40
50
Re
lati
v
118.11 121.12116.11
114.11119.11
112.09
Cells, tissue, fluidsAdds stepsNo MS technical variabilityQuantification at MS/MS level
9
Protein ExtractGels
Cells
Labeling Workflows
Tissue or Fluid
Gel matching
DIGE
SILAC
18O-Labeling ICAT TMTiTRAQ
Proteolysis
Radiolabeling
g
Peak Area
iTRAQFractionation
MS
MS2Peptide/Protein ID
ConfirmationMS3
Report Ion Intensity
AutoradiographyFluorescence
Changes in Protein Levels
(biomarkers and pathways)(biomarkers and pathways)
Phospholipid Growth Factor (S1P) on Pulmonary Endothelial Permeability
1
1.2
1.4
1.6
Re
sis
tan
ce
S1P
Resistance = Permeability
0
0.2
0.4
0.6
0.8
0 50 100 150 200 250 300
Time (min)
No
rma
lize
d R
Lipid Rafts?
Proteins involved?
Resistance = Permeability
Guo Y et al (2007) Mol Cell Proteomics 6:689
80
85
90
95
100
105
110
115459.31
291.23y1
y3
10
20
115.11
114.11
MRP_HumanGDVTAEEAAGASPAK (3+)
80
85
90
95
100
105
110
115459.31
291.23y1
y3
10
20
115.11
114.11
GDVTAEEAAGASPAK (3+)
iTRAQ Revealed Proteins More Abundant in S1P Stimulated Lipid Rafts
MRP Peptide Fragmentation
Co
ntr
ol
114 S
1P
115
A. Anti-MARCKS
Sample 1 Sample 2
Control S1P Control S1P
A. Anti-MARCKS
Sample 1 Sample 2
Control S1P Control S1P
Western Blots Confirm iTRAQ
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Intensity, counts
362.27 423.72230.17
674.42 717.39546.36
259.10
416.24 588.33115.11 846.42200.11
317.17241.10102.06 494.76
273.69523.28 745.46330.15 570.32
b1202.13
b2
b3
517.27
b5
b6
b7
b8917.43 b9
988.47 b101045.49
y2
y4
y5617.37
y6
y7
y32+
y42+
b72+
b92+
b92+
b4
114.0 115.0 116.0 117.0m/z, amu
0
10
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Intensity, counts
362.27 423.72230.17
674.42 717.39546.36
259.10
416.24 588.33115.11 846.42200.11
317.17241.10102.06 494.76
273.69523.28 745.46330.15 570.32
b1202.13
b2
b3
517.27
b5
b6
b7
b8917.43 b9
988.47 b101045.49
y2
y4
y5617.37
y6
y7
y32+
y42+
b72+
b92+
b92+
b4
114.0 115.0 116.0 117.0m/z, amu
0
10
MRP is up-regulated using both methods
C. Anti-MRP
D. Anti-Caveolin-1
B. Anti-p-MARCKS
C. Anti-MRP
D. Anti-Caveolin-1
B. Anti-p-MARCKS
Guo Y et al (2007) Mol Cell Proteomics 6:689
10
MRP siRNA Attenuates S1P Stimulated Endothelial Barrier Enhancement
1
1.2
1.4
1.6
sis
tan
ce
S1PS1P+Scrambled MRP S1P+Scrambled MRP siRNAsiRNA
S1P+MRP S1P+MRP siRNAsiRNA
MRPMRP siRNAsiRNA
0
0.2
0.4
0.6
0.8
0 50 100 150 200 250 300
Time (min)
No
rma
lize
d R
es
MRP MRP siRNAsiRNAoror
Scrambled MAP Scrambled MAP siRNAsiRNA
Guo Y et al (2007) Mol Cell Proteomics 6:689
Changes in Protein Modifications
(structure/function)(structure/function)
(acetylation, glycosylation, phosphorylation, nitrosation,
ubiquitination and novel cleavage sites)
Quantitative Phospho-Proteomics Workflow
Protein samples (1 mg per sample)
Peptides
iTRAQ Labeling
Phospho-Peptide enrich (Ti02)LCMS/MS Analysis
5% 95%
LCMS/MS Analysis
Protein ProfilingProtein present
Changes in Protein expression
Phospho-Peptide ProfilingPhosphorylations sites
Changes in Phosphorylation site
Distinguish site occupancy vs protein expression changes
iTRAQ PhosphoProteome Study (1% FDR)
5% BEFORE TiO2
enrichment
95% AFTER TiO2
enrichmentProtein groups identified 2691 2247Phosphoprotein groups identified 326 2134
Gut Epithelial Cell Cultures(+/-Kinase stimulated and Kinase knockout)
Unique Peptide Sequences 13876 5342Unique Peptide Sequences Containing Phosphorylation
445 4992
Peptides with pSer 338 4520Peptides with pThr 63 625Peptides with pTyr 6 28
Mark Donowitz
11
Kinetics
(protein turnover modification dynamics)(protein turnover, modification dynamics)
Which peptide is a better substrate?
Experimental Design:
T i ( id )
-Wade Gibson
Can we adapt iTRAQ reagents for kinetics experiments?
-Bob Cole
Two timecourses (one per peptide)
Two iTRAQ experiments (one for each timecourse)
Substrate and products labeled with same iTRAQtag at each timepoint
Different iTRAQ label for different timepoints
Labeling Scheme for Each Time Course
Time (min) iTRAQ Label0 1135 114
15 11530 11645 11760 11890 119
120 121
Mix all iTRAQ labeled substrates and proteins from all timepoints from one timecourse.
Run one MS analysis (LCMS/MS) for each timecourse.
Peptide 1
0
100
200
300
400
500
600
700
800
900
1000
Substrate 1
60
80
100
120
140
Product 1a
Peptide 2
0
10
20
30
40
50
60
70
80
90
100
300
400
500
600
700
800
900
Substrate 2
Product 2a
113 114 115 116 117 118 119 121
0 5 15 30 45 60 90 120
0
20
40
0
50
100
150
200
250
Product 1b
0
100
200
300
0
1
2
3
4
5
6
7
8
113 114 115 116 117 118 119 121
0 5 15 30 45 60 90 120
Product 2b
iTRAQ LabelTime (min)
12
Protein ExtractGelsDIGE
Gel matching
Cells Tissue or FluidSILAC
Proteolysis TMTiTRAQ
Relative Quantitative Proteomics Workflows
18O-Labeling ICAT
Radiolabeling
Spectral Counting
g iTRAQFractionation
MS
MS2
Peak AreaPeptide/Protein ID
ConfirmationMS3
AutoradiographyFluorescence
Report Ion Intensity
Densitometry
Quantitative Proteomics Methods
Label-Free Labeling
ChemicalMetabolic Enzymatic
DIGE (Difference Gel Electrophoresis)Radiolabeling 18O-Labeling
Relative Absolute
ICAT (Isotope-Coded Affinity Tags)
TMT (Tandem Mass Tags)
iTRAQ (Isobaric Tags for Relativeand Absolute Quantitation)
SILAC
Gel matchingDensitometryPeak IntensitySpectral Counting
AQUA (Absolute Quantification)MRM (Multiple Reaction Monitoring)QconCAT (Quantification Concatamers)PSAQ (Protein Standard Absolute Quantification)SWATH
Absolute Quantification (AQUA)by spiking in a Peptide standard
Protein A to quantify in sample
Selects FragmentsQuantifies
Triple Quadrupole MS
Spike
Heavy Stable Isotope Standard Peptide from Protein A
Meng and VeenstraJ. Proteomics 2011
AQUA PeptidesChoose (software can help)One or more peptides from a previously identified protein Must: Be easily detected by MS
Yield good fragmentation spectraHave an unique sequenceContain no modifiable amino acids (no M,C,S,T,Y,K,R etc)
SynthesizePeptides with stable isotopes (e g 13C 15N etc ) amino acidsPeptides with stable isotopes (e.g., C, N, etc.) amino acids
Standard CurvesCreate standard curves (Intensity vs amount) of synthetic peptide
Spike Stable isotope labeled peptides is spiked into protein sample
Compare peak intesities or areasNative peptide intensities to synthetic peptides Calculate amount from synthetic peptide standard curves
13
Increases sensitivity and signal to noise ratio
Spray
HPLC
Peptide MassSelection
PeptideFragmentation
Fragment MassSelection
Fragment MassSpectrum
Inte
ns
ity
m/zQ3Q2Q1
609/969
969609
Selective Reaction Monitoring (SRM)
Multiple Reaction Monitoring (MRM)
Increases sensitivity and signal-to-noise ratioIncrease chance of detecting in protein extractsSpike stable isotope peptide to quantifyMass accuracy very helpful Many fragment ions
from many peptidesWide (e.g 20 amu) peptide mass selection window
SWATH
Quantification Concatamers (QconCAT)
Quantify known proteins in a complex sampleCombines AQUA and MRM strategiesControls for protein digestion Validates other quantification methodsAlternative to western blotting
Nat Meth 2:585, 2005Nat Protocols 1:1029, 2006Mol Cell Proteomics 6:1416, 2007
Protein Extract
Cells
Protein Standard Absolute Quantification Workflow
Tissue or Fluid
Protein Fractionation
RecombinantHeavy Stable Isotope Standard of Protein to Quantify
Protein Standard Absolute Quantification (PSAQ)
Peak Area
Proteolysis
Peptide Fractionation
MSPeptide/Protein ID
MS2
Protein Extract
Cells
Protein Standard Absolute Quantification Workflow
Tissue or Fluid
Protein FractionationQconCat Chimeras
PSAQ Proteins
Absolute Quantitative Proteomics Workflows
Peak Area
Proteolysis
Peptide Fractionation
MSPeptide/Protein ID
MS2
AQUA Peptides
14
PSAQ ProteinsSlope = 1.05
QconCat ChimerasSlope = 0.58
AQUA PeptidesSlope = 0.21
Accuracy of Absolute
Quantification Workflows
Meng and VeenstraJ. Proteomics 2011
PSAQ ProteinsSlope = 1.08
AQUA PeptidesSlope = 0.0.4
QconCat ChimerasSlope = 0.19
Quantitative Proteomics Methods
Label-Free Labeling
ChemicalMetabolic Enzymatic
DIGE (Difference Gel Electrophoresis)Radiolabeling 18O-Labeling
Relative Absolute
ICAT (Isotope-Coded Affinity Tags)
TMT (Tandem Mass Tags)
iTRAQ (Isobaric Tags for Relativeand Absolute Quantitation)
SILAC
Gel matchingDensitometryPeak IntensitySpectral Counting
AQUA (Absolute Quantification)MRM (Multiple Reaction Monitoring)QconCAT (Quantification Concatamers)PSAQ (Protein Standard Absolute Quantification)SWATH
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