Quan%ta%on(with(XPRESS( and ASAPRa%o(sanjeeva/itpws/wp-content/uploads/... · 2016-01-30 · LectureOutline(• PrinciplesofquantaveproteomicsusingLCESIG MS/MS(• Pep%de(and(Protein(Quan%ta%on(with(XPRESS(

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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


•  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





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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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





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


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• Select “RUN XPRESS” • Select “RUN ASAPRatio”


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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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• Interface for protein ratio

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•  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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Individual peptides

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• Details on individual peptides

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• Interface for peptide ratio

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•  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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•  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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Sort by p values first and verify potentially interesting data

Identify and verify troublesome unique peptide ratios


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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


•  PepBde and Protein QuanBtaBon with XPRESS –  Running XPRESS –  Looking at results

•  PepBde and Protein QuanBtaBon with ASAPRaBo –  Running ASAPRaBo –  Looking at results

•  Exercises 61

Documents

Quan%ta%on(with(XPRESS( and ASAPRa%o(sanjeeva/itpws/wp-content/uploads/... · 2016-01-30 · LectureOutline(• PrinciplesofquantaveproteomicsusingLCESIG MS/MS(• Pep%de(and(Protein(Quan%ta%on(with(XPRESS(