Quan%ta%on with XPRESS. and. ASAPRa%o

Transcription

1 Quan%ta%on with XPRESS and ASAPRa%o 1

2 Pep%de and Protein Quan%ta%on Raw Mass Spec Data Pep%de Iden%fica%on Pep%de Valida%on Quan%ta%on Protein Assignment Protein List msconvert X!Tandem SpectraST SEQUEST* Mascot* Pep%deProphet iprophet PTMProphet ASAPRa%o XPRESS Libra ProteinProphet SBEAMS PIPE2 mzml pepxml protxml

3 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

4 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 Denatured protein complex Peptides RP chromatographic separation of peptides Identify proteins in complex Db search Mass Spec 4

5 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 5

6 Reversed- Phase Chromatography Reproducible: but a few erra%c data points may exist Tapered frit! - 2 to 4 kv! UV detector HPLC Peek microcross! Analytical Column (100 µm i.d.)! Grounded! waste close Precolumn! Peptide eluting profiling 6-way Divert Valve time 6

7 Electrospray Ioniza%on Mul%ple charge states: from +1 to +4 M + z H + = M(H + ) z m/z = (M+z*H)/z LC +1 ESI + HV - time

8 ESI- Tandem Mass Spectrometry identified peptides quantify peptides MS MS/MS (CID) identify peptides 8

9 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!!!!!! y4 y5 y6 y7 y8 y9 y10 y11 y12 y13 y14 y15 y16 y17 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 A D A T L S Q I T N N I D b

10 Single Ion Chromatogram 2D view: m/z, intensity 3D view: m/z, intensity, time MS scans intensity intensity m/z Single Ion Current (SIC) Trace intensity time (scan #) m/z= scan # 10

11 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 11

12 Different Labeling Methods Metabolic labeling 13 C, 15 N, SILAC Chemical reac%on ICAT, cleavable ICAT itraq Enzyme reac%on 18 O 12

13 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 MS 2 spectrum to be quanbfied Accuracy: ±10-30% Dynamic range: ~100 fold TPP provides 2 op%ons: Xpress and ASAPRa%o 13

14 Protein Iden%fica%on and Quan%fica%on Hierarchy Structure protein VNG0679G peptide GCPTAELRFDDMR LGDKGCPTAELR LC peak haloicat2_33 (scan 1274) haloicat2_32 (scan 1306) haloicat2_33 (scan 1024) CID heavy, +2 heavy, +2 light, +2 light, +3 heavy, +3 Peptide IDs & Ratios Protein IDs & Ratios 14

15 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

16 XPRESS Publica%on Han DK, Eng J, Zhou H, and Aebersold R. (2001) Nature Biotechnology 19:

17 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 17

18 XPRESS Protein Ra%o Calculated as the Geometric Mean of the cons%tuent pep%de ra%os Uncertainty is also calculated 18

19 Running XPRESS: Petunia Interface 19

20 Running XPRESS: Command- line Use the X flag for xinteract 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 -F<num> partner 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 20

21 XPRESS Pep%deProphet Results 21

22 XPRESS ProteinProphet Results 22

23 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 23

24 Defini%ons protein Protein Ratio VNG0679G Unique peptide ratio peptide GCPTAELRFDDMR LGDKGCPTAELR LC peak haloicat2_33 (scan 1274) haloicat2_32 (scan 1306) haloicat2_33 (scan 1024) CID heavy, +2 heavy, +2 light, +2 light, +3 heavy, +3 Peptide (CID) ratio 24

25 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

26 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 26

27 Example on Single- Ion Chromatogram Red: raw Blue: fitting Green: area Pink: background T-bar: CID 27

28 Pep%de Charge Distribu%on Out of 1857 peptides

29 Single-Ion Chromatogram of +2 Ion 29

30 Single-Ion Chromatogram of +3 Ion 30

31 Single-Ion Chromatogram of +4 Ion 31

32 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 32

33 Example on Peptide Ratio mean +- SD (CV%) CV = SD/mean SD: Std.Dev, CV: Coeff. Of Variation 33

34 Evalua%on of Unique Pep%de Abundance Ra%o protein VNG0679G Unique peptide ratio peptide GCPTAELRFDDMR LGDKGCPTAELR LC peak Step 2 haloicat2_33 (scan 1274) haloicat2_32 (scan 1306) haloicat2_33 (scan 1024) CID heavy, +2 heavy, +2 light, +2 light, +3 heavy, +3 Step 1 Peptide ratio 34

35 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 Step 1 35

36 Evalua%on of Unique Pep%de Ra%o Step 2 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 36

37 Example of Unique Peptide Ratio Step 2 Step 1 Step 1 37

38 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 38

39 Example on Protein Ratio 0.54 (outlier)

40 Sample- Dependent Ra%o Normaliza%on To Correct Systematic Error Due to Sample Handling

41 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 41

42 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) 42

43 ASAPRatio Main Features 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 43

44 How to Use TPP for Data Analysis in Quan%ta%ve Proteomics Start TPP Click on Analyze Peptides Select the xml files that you want to analyze Same as when running PeptideProphet 44

45 How to Use TPP for Data Analysis in Quan%ta%ve Proteomics Select RUN XPRESS Select RUN ASAPRatio 45

46 How to Use TPP for Data Analysis in Quan%ta%ve Proteomics interact.prot.xml 46

47 How to Interpret ASAPRa%o Results 47

48 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 48

49 How to Interpret ASAPRatio Results Interface for protein ratio 49

50 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 50

51 How to Interpret ASAPRatio Results Individual peptides 51

52 How to Interpret ASAPRatio Results Details on individual peptides 52

53 How to Interpret ASAPRatio Results Interface for peptide ratio 53

54 How to Interpret ASAPRatio Results 54

55 How to Interpret ASAPRatio Results 55

56 How to Interpret ASAPRatio Results 56

57 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 57

58 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 58

59 How to Interpret ASAPRa%o Results Sort by p values first and verify potentially interesting data Identify and verify troublesome unique peptide ratios 59

60 How to Interpret ASAPRa%o 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 60

61 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