Protein Valida-on (Sta-s-cal Inference) and Protein Quan-fica-on. Center for Mass Spectrometry and Proteomics Phone (612) (612)

Transcription

1 Protein Valida-on (Sta-s-cal Inference) and Protein Quan-fica-on

2 Terminology Pep-de Spectrum Match Target / Decoy False discovery rate Shared pep-de Parsimony One hit wonders

3 SPECTRUM Rela-ve Abundance m/z 3

4 Tandem mass spectrometry and pep/de sequence y-series: b-series: y 14 H 2 N-N--S--G--D--I--V--N--L--G--S--I--A--G--R-COOH b 1 y 13 y 12 y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2 y 1 b 2 b 3 b 4 b 5 b 6 b 7 b 8 b 9 b 10 b 11 b 12 b 13 b 14 Relative Abundance m/z An MS/MS spectrum contains a mixture of b and y ions 4

5 PEPTIDE SPECTRUM MATCH Mass spectrum Pep/de Spectral Match Reference Protein Database from genomic annotation

6 PEPTIDE SPECTRUM MATCH Visual inspection Scoring function f(s,p) measures the quality of the match between spectrum S and peptide sequence P Look at fragment ions predicted mass expected intensity compare to next best presence of immonium ions etc. Nesvizhskii et al Nature Methods - 4, (2007) 6

7 PEPTIDE SPECTRAL MATCH Nesvizhskii et al Nature Methods - 4, (2007) 7

8 TARGET-DECOY SEARCH

9 TARGET-DECOY SEARCH INPUTS : PEAKLISTS and SEARCH db SEARCHGUI PEPTIDESHAKER 9

10 PROTEIN INFERENCE: FROM PEPTIDES TO PROTEINS Slide from Alexey Nesvizshkii talk at hwp:// 10

11 Nesvizhskii, A. I.; Aebersold, R. Mol. Cell. Proteom. 4.10, , 2005 Shared Pep-des

12 Shared Pep-des YMACCLLYR SIQFVDWCPTGFK Tubulin alpha 6 Tubulin alpha 4 Tubulin alpha 3 85% 85% 85%

13 AEPTIR 85% From Pep-des to Proteins IDVCIVLLQHK 65% Protein (4%) NTGDR 25% 0.15 * 0.35 * 0.75 = 0.04 Feng J, Naiman DQ, Cooper B. Anal Chem May 15;79(10):

14 One Hit Wonders IDVCIVLLQHK 95% Protein (5%)

15 One Hit Wonders Quan-fy as a score: If different pep-des agree: Good! If pep-des are one- hit- wonders: Bad! Protein Prophet, etc. Then can use FDR at protein level

16 Pep-de / Protein Quan-fica-on

17 Terminology Absolute Quan-fica-on Rela-ve Quan-fica-on Label- free Normaliza-on Labelled itraq Reporter ions

18 Pep-de / Protein Quan-fica-on Absolute Es-mate the molar amount of protein / pep-de in the biological sample PTMs Valida-on Rela-ve Fold change / sta-s-cally significant difference between 2 biological states Biological varia-on Biomarker studies

19 Label- free Area Under Curve MS1 Integrate XIC Spectral Coun-ng MS2 High abundant proteins Käll L, Vitek O (2011) Computa-onal Mass Spectrometry Based Proteomics. PLoS Comput Biol 7(12): e doi: /journal.pcbi

20 MS Quan-fica-on MS not inherently quan-ta-ve Physiochemical proper-es invoke different MS responses MS only samples a small percentage of total pep-des Bias and variability Population Biological Instrument Sample Handling

21 Normaliza-on Remove bias and variability between runs Global commonly used Median scale Total ion current (TIC) Local very recent development Proximity- based intensity normaliza-on (PIN)

22 Labeled Quan-fica-on Run samples simultaneously on in a single run Add label to samples Mix samples together Compute ra-os / sta-s-cally significant diffs.

23 Labeled Isobaric MS2, itraq Number of samples Synthe-c Pep-des MS1 Absolute (AQUA) Metabolic MS1, SILAC Not higher life forms Käll L, Vitek O (2011) Computa-onal Mass Spectrometry Based Proteomics. PLoS Comput Biol 7(12): e doi: /journal.pcbi

24 itraq 8- Plex Reagent Chemical Structure Isobaric Tag Total mass = 305 Reporter Group , 121 m/z N N ~~~~~~~ O N O Amine specific pep/de reac/ve group (NHS) N- hydroxysuccinimide O Balance Group (?) Mass 184, m/z Isobaric Reporter Groups , 121 m/z N N N N N+ N+ N+ N CH 3 13 CH 3 13 CH 3 N 15 N 15 N 15 N 15 N+ 15 N+ 15 N+ CH 2 15 N Applied Biosystems has granted permission to use this slide.

25 itraq Experiment Obtain protein- containing sample, extract protein Normal Brain Tissue- 1 Normal Brain Tissue- 2 Huntington s Disease-1 Huntington s Disease-2 Reduce, alkylate Cysteines Reduce, alkylate Cysteines Reduce, alkylate Cysteines Reduce, alkylate Cysteines Proteoly-c Diges-on Trypsin Digest Trypsin Digest Trypsin Digest Trypsin Digest Label pep-des with itraq Reagents itraq TAG 114 itraq TAG 115 itraq TAG 116 itraq TAG 117 MIX 2D LC- MS/MS Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695

26 itraq Experiment MS2 Spectrum Intensity, counts Intensity, counts +TOF Product (513.3): Experiment 2, min from keyxx001_perez061_fxn13_queb2_1004.wiff a= e-004, t0= e y a b b1 y9(+2) y y a b Pep-de itq VAIVVGAPR MWPeptide mono = VAIVVGAPR Protein MW Match = Platelet Protein ID = platelet membrane glycoprotein glycoprotein 11b 11b a4 y y6 a Max counts. Reporter Ion Mass Tags from which quantitation is calculated Pep-de match is made from product ions, e.g., b- and y- ion series 300 y9 y2 y I 70.1 I I b y m/z, amu y9

27 itraq Results Reporter ion intensi-es reflect rela-ve pep-de amounts control disease % Intensity No change disease:control Mass (m/z)

28 itraq Results What fold changes are significant? Do they represent biological relevance as opposed to experimental variability? 100 control disease E Increase disease:control % Intensity Mass (m/z) control disease Decrease disease:control % Intensity Mass (m/z)

29 Labeled & Label- free From Käll L, Vitek O (2011) Computa-onal Mass Spectrometry Based Proteomics. PLoS Comput Biol 7(12): e doi: / journal.pcbi