isotopic-labeling and mass spectrometry-based quantitative...

Isotopic-Labeling and Mass Spectrometry-Based Quantitative

ProteomicsXiao-jun Li, Ph.D.

Current address: Homestead ClinicalDay 4

October 19, 2006

Protein Quantification

TPPTPP

xINTERACTxINTERACT

PeptideProphetPeptideProphet XPRESS/ASAPRatioLibra

XPRESS/ASAPRatioLibra

mzXML file formatmzXML file format

ProteinProphetProteinProphet

SBEAMSSBEAMS

PeptideAtlasPeptideAtlas

Pep3DPep3DSEQUEST/COMETMascot/ProbID/SpectraST

SEQUEST/COMETMascot/ProbID/SpectraST

CytoscapeCytoscape

LC-MS/MS DataLC-MS/MS Data

pepXML file formatpepXML file format

protXML file formatprotXML file format

QualscoreQualscore

Gaggle…Gaggle…

XLinkXLink

• Principles of quantitative proteomics using LC-ESI-MS/MS

• Introduction to ASAPRatio: software tool for quantitative proteomics using isotopic labeling

Outline

Summary of LC-ESI-MS/MS• Protein mixtures are digested into peptides• Peptides are concentrated and fractionated by separation technologies

such as SCX, IEF, RP, etc.• While eluting from RP column, peptides are ionized by ESI and analyzed

by MS/MS • Peptides are identified from CID spectra• Peptides are usually quantified from MS signatures

– Except in the case of iTRAQ

RP

Identify proteinsin complex

chromatographicseparation of peptidesDenatured protein

complexPeptides

Mass SpecDb search

Complications

Shotgun MS detects peptides not proteins– Multiple peptides per protein– Multiple proteins per peptide

Strong Cation-Exchange Chromatograph– Fair but not great separation power– Same peptide separated into several fractions

Reversed-Phase ChromatographyReproducible: but a few erratic data points may exist

time

Analytical Column (100 μm i.d.)

Grounded

Peek micro-cross

Tapered frit

Precolumnwaste

6-way Divert Valve

close

- 2 to 4 kV

Peptide eluting profiling

% A

cN%

AcNR

.A.(%

)R

.A.(%

)

00

5050

100100

00

100100

5050

Time (min)Time (min)1010 2020 4040 5050 6060 70703030

HPLCUV detector

Electrospray Ionization

• Multiple charge states: from +1 to +4

M + z H+ = M(H+)z m/z = (M+z*H)/z

HV+ -

LC

time

ESI

+4+3

+2

+1

ESI-Tandem Mass Spectrometry

time

9 ions; 3 peptides; multiple ions/peptide (different charge states)

Peptide Identification• Match CID (MS/MS) spectra with database

– SEQUEST, MASCOT, X!Tandem, …

• Multiple IDs for the same peptide – different isotopes: light and heavy– different charge states: +1, +2, +3 – repeating IDs: same isotope and same charge state

m/zm/z400400 800800 12001200 16001600 20002000

00

5050

100100 y9 y12y4 y8y5 y11 y14 y15 y17y13 y16y7y6 y10

b15b4 b10 b14b7 b8b6b3b2 b5 b9 b11 b12 b13

R.A

.(%)

R.A

.(%) D SQTNI N I AL T D A

A SLTAD NIQ NT DI

600600 800800 10001000 12001200 14001400600600 800800 10001000 12001200 14001400

Single Ion Chromatogram

m/z

inte

nsity2D 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

m/zm/z

m/zm/z

1000.2

Single-Ion Chromatogram

time

time

Peptide Quantitation• Area under SIC is proportional to peptide abundance• PROBLEM

Ionization efficiency of each peptide is different– Depends on the peptide molecular properties (e.g. number of

basic residues)

• ONE SOLUTIONSamples labeled with different stable isotopes

• Chemically identical• Peptides are identified before quantification• Distinguishable by MS in mass shift• Peptide abundance ratio measured by ratio of SIC areas

Different Labeling Methods

• Metabolic labeling 13C, 15N, SILAC

• Chemical reaction ICAT, cleavable ICAT iTRAQ

• Enzyme reaction 18O

Summary of Quantitative LC-MS/MS Approach

• Samples are isotopically labeled• Simultaneously identify & quantify thousands of

proteins in complex samples• Accuracy: ±20-30%• Dynamic range: ~100 fold

• Principles of quantitative proteomics using LC-ESI-MS/MS

• Introduction to ASAPRatio: software tool for quantitative proteomics using isotopic labeling

Outline

How to Use TPP for Data Analysis in Quantitative Proteomics

•Start TPP•Click on “Analyze Peptides”•Select the xml files that you want to analyze•Same as when running PeptideProphet

•Select “RUN PeptideProphet”•Select “RUN ProteinProphet afterwards”



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


•Click on “RUN XInteract”•Wait until “Command Status” turns orange•Click to view output files•View “interact-prot.shtml” file

•interact-prot.shtml


How to Interpret ASAPRatioResults


Protein ratio and its standard deviation

Number of unique peptides

Normalized protein ratio and its standard deviation

Protein p-value for differential expression


•Interface for protein ratio


•Protein profiling based on their ratios•Normalized ratio: r* = r/r0•P-value: significance in differential expression; how far is the data from r0


Individual peptides


•Details on individual peptides


•Interface for peptide ratio


•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


•Sort by p values first and verify potentially interesting data

•Identify and verify troublesome unique peptide ratios


•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”

• Able to handle various labeling methods (except iTRAQ) • Estimate error on peptide and protein ratios• Calculate peptide ratios from multiple charge states

– Not just from charge state in which the CID was matched

• Chromatogram signal background subtraction to increase the dynamic range

• Calculate protein ratios by using connection of peptides to proteins computed by ProteinProphet

• Evaluate p-value for protein profiling• Detect outliers: Dixon’s test• Easy to use user interface for manual validation of ratios

ASAPRatio Main Features

Summary

• Quantitative LC-ESI-MS/MS is a powerful method for identifying and quantifying proteins in complex samples

• ASAPRatio provides a user-freindly platform for analyzing LC-ESI-MS/MS data

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