Isotopic-Labeling and Mass Spectrometry-Based Quantitative Proteomics Xiao-jun Li, Ph.D. Current address: Homestead Clinical Day 4 October 19, 2006
Protein Quantification LC-MS/MS Data XLink mzxml file format TPP SEQUEST/COMET Mascot/ProbID/SpectraST Pep3D Qualscore pepxml file format xinteract PeptideProphet XPRESS/ASAPRatio Libra ProteinProphet protxml file format Gaggle SBEAMS Cytoscape PeptideAtlas
Outline Principles of quantitative proteomics using LC- ESI-MS/MS Introduction to ASAPRatio: software tool for quantitative proteomics using isotopic labeling
Outline Principles of quantitative proteomics using LC-ESI-MS/MS Introduction to ASAPRatio: software tool for quantitative proteomics using isotopic labeling
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 Denatured protein complex Peptides RP chromatographic separation of peptides Identify proteins in complex Db search Mass Spec
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 Chromatography Reproducible: but a few erratic data points may exist Tapered frit Peek microcross -2 to 4 kv Analytical Column (100 μm i.d.) Grounded UV detector R.A.(%) 100 50 0 HPLC 0 10 20 30 40 50 60 70 Time (min) 100 50 % AcN waste close Precolumn Peptide eluting profiling 6-way Divert Valve time
Electrospray Ionization Multiple charge states: from +1 to +4 M + z H + = M(H + ) z m/z = (M+z*H)/z LC +1 ESI + HV - time +2 +3 +4
ESI-Tandem Mass Spectrometry 9 ions; 3 peptides; multiple ions/peptide (different charge states) time
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 600 800 1000 1200 1400 R.A.(%) 100 y4 y5 y6 y7 y8 y9 y10 y11 y12 y13 y14 y15 y16 y17 50 D I N N T I Q S L T A D A b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 A D A T L S 0 400 800 1200 1600 2000 m/z Q I T N N I D
Single Ion Chromatogram intensity intensity intensity 2D view: m/z, intensity Single Ion Current (SIC) Trace m/z 3D view: m/z, intensity, time m/z m/z time (scan #) m/z=1000.2 MS scans m/z m/z 1000.2 scan #
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 SOLUTION Samples 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 13 C, 15 N, SILAC Chemical reaction ICAT, cleavable ICAT itraq Enzyme reaction 18 O
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
Outline Principles of quantitative proteomics using LC-ESI-MS/MS Introduction to ASAPRatio: software tool for quantitative proteomics using isotopic labeling
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
How to Use TPP for Data Analysis in Quantitative Proteomics Select RUN PeptideProphet Select RUN ProteinProphet afterwards
How to Use TPP for Data Analysis in Quantitative Proteomics Select RUN XPRESS Select RUN ASAPRatio
How to Use TPP for Data Analysis in Quantitative Proteomics Click on RUN XInteract Wait until Command Status turns orange Click to view output files View interact-prot.shtml file
How to Use TPP for Data Analysis in Quantitative Proteomics interact-prot.shtml
How to Interpret ASAPRatio Results
How to Interpret ASAPRatio Results Protein ratio and its standard deviation Protein p-value for differential expression Number of unique peptides Normalized protein ratio and its standard deviation
How to Interpret ASAPRatio Results Interface for protein ratio
How to Interpret ASAPRatio Results Protein profiling based on their ratios Normalized ratio: r* = r/r 0 P-value: significance in differential expression; how far is the data from r 0
How to Interpret ASAPRatio Results Individual peptides
How to Interpret ASAPRatio Results Details on individual peptides
How to Interpret ASAPRatio Results Interface for peptide ratio
How to Interpret ASAPRatio Results
How to Interpret ASAPRatio Results
How to Interpret ASAPRatio Results
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 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 ASAPRatio Results Sort by p values first and verify potentially interesting data Identify and verify troublesome unique peptide ratios
How to Interpret ASAPRatio Results 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
ASAPRatio Main Features 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
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