Quan%ta%on(with(XPRESS( and ASAPRa%o(sanjeeva/itpws/wp-content/uploads/... · 2016-01-30 ·...
Transcript of Quan%ta%on(with(XPRESS( and ASAPRa%o(sanjeeva/itpws/wp-content/uploads/... · 2016-01-30 ·...
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Quan%ta%on with XPRESS and
ASAPRa%o
Pep%de and Protein Quan%ta%on
Quan%ta%on Raw Mass Spec Data
msconvert
Pep%de Iden%fica%on
X!Tandem
SpectraST
SEQUEST*
Mascot*
Pep%de Valida%on
Pep%deProphet
iProphet
PTMProphet
Protein Assignment
ProteinProphet
Protein List
SBEAMS
PIPE2
pepXML protXML mzML
ASAPRa%o
XPRESS
Libra
Lecture Outline
• Principles of quan%ta%ve proteomics using LC-‐ESI-‐MS/MS
• Pep%de and Protein Quan%ta%on with XPRESS – Running XPRESS – Looking at results
• Pep%de and Protein Quan%ta%on with ASAPRa%o – Running ASAPRaBo – Looking at results
• Exercises 3
Summary of LC-‐ESI-‐MS/MS
• Protein mixtures are digested into pep%des • Pep%des are concentrated and frac%onated by separa%on technologies such
as SCX, IEF, RP, etc.
• While elu%ng from RP column, pep%des are ionized by ESI and analyzed by MS/MS
• Pep%des are iden%fied from CID spectra
• Pep%des are usually quan%fied from MS signatures – Except in the case of iTRAQ
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RP
Identify proteins in complex
chromatographic separation of peptides Denatured protein
complex Peptides
Mass Spec Db search
Complica%ons
Shotgun MS detects pep%des not proteins – MulBple pepBdes per protein
– MulBple proteins per pepBde
Strong Ca%on-‐Exchange Chromatograph – Fair but not great separaBon power
– Same pepBde separated into several fracBons
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Reversed-‐Phase Chromatography
Reproducible: but a few erra%c data points may exist
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time
Analytical Column (100 µm i.d.)!
Grounded!
Peek micro-cross!
Tapered frit!
Precolumn!waste
6-way Divert Valve
close
- 2 to 4 kV!
Peptide eluting profiling
HPLC UV detector
Electrospray Ioniza%on
Mul%ple charge states: from +1 to +4
M + z H+ = M(H+)z m/z = (M+z*H)/z
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HV + -
LC
time
ESI
+4 +3
+2
+1
ESI-‐Tandem Mass Spectrometry
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MS
MS/MS
(CID)
identify peptides
quantify peptides +2
+3
+3
+2
identified peptides
Pep%de Iden%fica%on
• Match CID (MS/MS) spectra with database – SEQUEST, MASCOT, X!Tandem, …
• Mul%ple IDs for the same pep%de – different isotopes: light and heavy – different charge states: +1, +2, +3 – repeaBng IDs: same isotope and same charge state
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y9 y12 y4 y8 y5 y11 y14 y15 y17 y13 y16 y7 y6 y10
b15 b4 b10 b14 b7 b8 b6 b3 b2 b5 b9 b11 b12 b13 D S Q T N I N I A L T D A
A S L T A D N I Q N T D I
600 600 800 800 1000 1000 1200 1200 1400 1400 600 600 800 800 1000 1000 1200 1200 1400 1400
!
!
!
!
! !
Single Ion Chromatogram
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m/z
inte
nsity
2D view: m/z, intensity
intensity
scan #
m/z=1000.2
Single Ion Current (SIC) Trace
3D view: m/z, intensity, time
MS scans
time (scan #) in
tens
ity
Pep%de Quan%ta%on
• Area under SIC is propor%onal to pep%de abundance • PROBLEM
Ioniza%on efficiency of each pep%de is different – Depends on the pepBde molecular properBes (e.g. number of basic
residues)
• ONE SOLUTION Samples labeled with different stable isotopes
• Chemically idenBcal
• PepBdes are idenBfied before quanBficaBon • DisBnguishable by MS in mass shi\
• PepBde abundance raBo measured by raBo of SIC areas
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Different Labeling Methods
• Metabolic labeling 13C, 15N, SILAC
• Chemical reac%on ICAT, cleavable ICAT iTRAQ
• Enzyme reac%on 18O
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Summary of Quan%ta%ve LC-‐MS/MS Approach
• Samples are isotopically labeled
• Simultaneously iden%fy & quan%fy thousands of proteins in complex samples – PepBde ion must be idenBfied in MS2 spectrum to be quanBfied
• Accuracy: ±10-‐30% • Dynamic range: ~100 fold
• TPP provides 2 op%ons: Xpress and ASAPRa%o 13
Protein Iden%fica%on and Quan%fica%on
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Hierarchy Structure
Peptide IDs & Ratios Protein IDs & Ratios
VNG0679G
heavy, +3
haloICAT2_32 (scan 1306) haloICAT2_33 (scan 1024)
LGDKGCPTAELR GCPTAELRFDDMR
haloICAT2_33 (scan 1274)
heavy, +2 light, +3 heavy, +2 light, +2
protein
peptide LC peak
CID
Lecture Outline
• Principles of quanBtaBve proteomics using LC-‐ESI-‐MS/MS
• Pep%de and Protein Quan%ta%on with XPRESS – Running XPRESS – Looking at results
• PepBde and Protein QuanBtaBon with ASAPRaBo – Running ASAPRaBo – Looking at results
• Exercises 15
XPRESS Publica%on
16 Han DK, Eng J, Zhou H, and Aebersold R. (2001) Nature Biotechnology 19:946-51.
XPRESS Pep%de Ra%o
• Calculated from SIC of charge state in which pep%de was iden%fied
• Smoothing done with a Buherworth low-‐pass filter
• No background es%ma%on
• Works with different labeling methods – ICAT, SILAC, etc
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XPRESS Protein Ra%o
• Calculated as the Geometric Mean of the cons%tuent pep%de ra%os
• Uncertainty is also calculated
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Running XPRESS: Petunia Interface
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Running XPRESS: Command-‐line
• Use the –X flag for xinteract
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xpressoptions [will run XPRESS analysis with any specified options that follow the 'X']: -m<num> change XPRESS mass tolerance (default=1.0) -l<str> change labeled residues (default='C') -n<str>,<num> change XPRESS residue mass difference for <str> to
<num> (default=9.0) -b heavy labeled peptide elutes before light labeled
partner -F<num> fix elution peak area as +-<num> scans (<num>
optional, default=5) from peak apex -L for ratio, set/fix light to 1, vary heavy -H for ratio, set/fix heavy to 1, vary light -M for metabolic labeling; ignore all other parameters,
assume IDs are normal and quantify w/corresponding 15N heavy pair
-N for metabolic labeling; ignore all other parameters, assume IDs are 15N heavy and quantify corresponding 14N light pair
XPRESS Pep%deProphet Results
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XPRESS ProteinProphet Results
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Lecture Outline
• Principles of quanBtaBve proteomics using LC-‐ESI-‐MS/MS
• PepBde and Protein QuanBtaBon with XPRESS – Running XPRESS – Looking at results
• Pep%de and Protein Quan%ta%on with ASAPRa%o – Running ASAPRa%o – Looking at results
• Exercises
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Defini%ons
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VNG0679G
heavy, +3
haloICAT2_32 (scan 1306) haloICAT2_33 (scan 1024)
LGDKGCPTAELR GCPTAELRFDDMR
haloICAT2_33 (scan 1274)
heavy, +2 light, +3 heavy, +2 light, +2
protein
peptide LC peak
CID
Peptide (CID) ratio
Unique peptide ratio
Protein Ratio
ASAPRa%o Methodology
• Reconstruc%on of single-‐ion chromatograms
• Evalua%on of pep%de abundance ra%os
• Evalua%on of unique pep%de abundance ra%os
• Evalua%on of protein abundance ra%os
• Sample-‐dependent ra%o normaliza%on
• Large-‐scale protein profiling
Anal. Chem.; 2003; 75(23) pp 6648-‐6657.
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Reconstruc%on of Single-‐Ion Chromatogram
• Assume pep%de iden%fica%on correct
• Raw chromatogram – Summarize MS intensiBes within a m/z window and trace the sum in
Bme
• Smooth chromatogram – Savitzsky-‐Golay smooth filter
• Subtract background and calculate area • Es%mate elu%on %me of isotopic partner
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Example on Single-‐Ion Chromatogram
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Red: raw
Blue: fitting
Green: area
Pink: background
T-bar: CID
Pep%de Charge Distribu%on
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Out of 1857 peptides
4 3
1
2
+1 +4 +3 +2
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Single-Ion Chromatogram of +2 Ion
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Single-Ion Chromatogram of +3 Ion
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Single-Ion Chromatogram of +4 Ion
Evalua%on of Pep%de Abundance Ra%o
• Evaluate a pep%de ra%o with error from each available charge state
• Use Dixon’s test to iden%fy any outliers • Weight charge states by chromatogram areas
• Use sta%s%cal methods to calculate pep%de ra%o and error
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Example on Peptide Ratio
mean +- SD (CV%)
CV = SD/mean
SD: Std.Dev, CV: Coeff. Of Variation
Evalua%on of Unique Pep%de Abundance Ra%o
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VNG0679G
heavy, +3
haloICAT2_32 (scan 1306) haloICAT2_33 (scan 1024)
LGDKGCPTAELR GCPTAELRFDDMR
haloICAT2_33 (scan 1274)
heavy, +2 light, +3 heavy, +2 light, +2
protein
peptide LC peak
CID
Peptide ratio
Unique peptide ratio
Step 1
Step 2
Evalua%on of Unique Pep%de Ra%o
• Group abundance ra%os of same pep%de and same RP elu%on peak together
– isotopic forms, charge states, repeats
• Most of them same
• If not: - Weight data points by their largest chromatogram areas
- Calculate mean and standard deviaBon
- Use Dixon’s test for outliers
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Step 1
• Group abundance ra%os of same pep%de but different RP elu%on peaks together
– SCX fracBons, RP eluBon Bmes
• Weight data points by their largest chromatogram areas
• Calculate mean and standard devia%on
• Use Dixon’s test for outliers
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Step 2
Evalua%on of Unique Pep%de Ra%o
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Example of Unique Peptide Ratio
Step 1
Step 1
Step 2
Evalua%on of Protein Abundance Ra%o
• Collect all unique pep%de ra%os of same protein together
• Use Dixon’s test on outliers – misidenBficaBon, modificaBon, etc.
• Weight data points by error
• Use sta%s%cal methods to calculate mean and standard devia%on
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Example on Protein Ratio
2.33
1.97 0.54 (outlier)
Sample-‐Dependent Ra%o Normaliza%on
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2.173+-0.683 0.865+-0.234
To Correct Systematic Error Due to Sample Handling
Sample-‐Dependent Ra%o Normaliza%on Condi%on: Background Proteins Dominant
• Fit log10(unique pep%de ra%o) with normal distribu%on (Fig. 5, ASAPRa%o paper)
• Normalize protein ra%os by peak ra%o
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Large-‐Scale Protein Profiling
• Evaluate p value for each protein p value: probability of a protein belonging to background group
• P value depends on: • Specify significance level (by user)
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• Able to handle various labeling methods (except iTRAQ)
• Es%mate error on pep%de and protein ra%os
• Calculate pep%de ra%os from mul%ple charge states – Not just from charge state in which the CID was matched
• Chromatogram signal background subtrac%on to increase the dynamic range
• Calculate protein ra%os based on pep%des that were assigned to proteins by ProteinProphet
• Evaluate p-‐value for protein profiling • Detect outliers: Dixon’s test • Easy to use user interface for manual valida%on of ra%os
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ASAPRatio Main Features
How to Use TPP for Data Analysis in Quan%ta%ve Proteomics
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• Start TPP • Click on “Analyze Peptides” • Select the xml files that you want to analyze • Same as when running PeptideProphet
How to Use TPP for Data Analysis in Quan%ta%ve Proteomics
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• Select “RUN XPRESS” • Select “RUN ASAPRatio”
How to Use TPP for Data Analysis in Quan%ta%ve Proteomics
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• interact.prot.xml
How to Interpret ASAPRa%o Results
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How to Interpret ASAPRatio Results
Protein ratio and its standard deviation
Number of unique peptides
Normalized protein ratio and its standard deviation
Protein p-value for differential expression
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How to Interpret ASAPRatio Results
• Interface for protein ratio
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How to Interpret ASAPRatio Results
• Protein profiling based on their ratios
• Normalized ratio: r* = r/r0 • P-value: significance in
differential expression; how far is the data from r0
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How to Interpret ASAPRatio Results
Individual peptides
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How to Interpret ASAPRatio Results
• Details on individual peptides
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How to Interpret ASAPRatio Results
• Interface for peptide ratio
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How to Interpret ASAPRatio Results
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How to Interpret ASAPRatio Results
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How to Interpret ASAPRatio Results
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How to Interpret ASAPRatio Results
• Changes can be made • Click “Evaluate Ratio”
for new results • Notice new interim ratio • If you like the changes,
click on “Interim Ratio” under “Set Accepted Ratio to” for record
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How to Interpret ASAPRatio Results
• Changes can be made • Click “Evaluate Ratio”
for new results • Notice new interim ratio • If you like the changes,
click on “Interim Ratio” under “Set Accepted Ratio to” for record
How to Interpret ASAPRa%o Results
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Sort by p values first and verify potentially interesting data
Identify and verify troublesome unique peptide ratios
How to Interpret ASAPRa%o Results
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For peptides of same experiment, verify one peptide ratio and reject others
Pay attention to unusual data: large error, 1:0, 0:1, or “unknown”
Lecture Outline
• Principles of quanBtaBve proteomics using LC-‐ESI-‐MS/MS
• PepBde and Protein QuanBtaBon with XPRESS – Running XPRESS – Looking at results
• PepBde and Protein QuanBtaBon with ASAPRaBo – Running ASAPRaBo – Looking at results
• Exercises 61