1. Hanyang Univ. Introduction to Mass Spectrometry-based Proteomics 1

2. Hanyang Univ. Central dogma of molecular biology transcription mRNADNAProtein translation 2

3. Hanyang Univ. Alternative Splicing ALTERNATIV E I AM THE FIRST ALTERNATIVE IAMTHEQSRTVFIRSTQPBASRTVSECONDQABDECTV exon1 exon2 exon3 exon4 intron1intron2intron3 IAMTHEFIRSTALTERNATIV E 3

4. Hanyang Univ. Alternative Splicing I AM THE SECOND ALTERNATIVE IAMTHEQSRTVFIRSTQPBASRTVSECONDQABDECTVALTERNATIV E exon1 exon2 exon3 exon4 intron1intron2intron3 IAMTHESECONDALTERNATIV E 4

5. Hanyang Univ. Proteomics Post-translational Modification (PTM) change in cellular localization protein-protein interaction Various Cellular Functions conformational change 5

6. Hanyang Univ. Genomics vs. Proteomics DNAProteins Easily amplified by PCRNo amplification technique Can be “unfolded” and “refolded” reversibly Folded structure very sensitive to conditions Chemically simple – negatively charged backbone, 4 nucleotides Chemically complex – 20 amino acids of non-polar, polar, charge, or hydrophobic character The amount of DNA is “stable” within a cell Protein concentration within a cell are highly variable DNA molecules tend not to associate Proteins often form complexes Blueprint vs. Machine 6

7. Hanyang Univ. Proteomics: study of protein Protein expression Protein structure Protein- protein interaction 7

8. Hanyang Univ. Proteomics http://www.childrenshospital.org/research/_proteomics /index.html 8

9. Hanyang Univ. Sample - Protein Digestion Protein  An amino acid sequence MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRKL HQEDNDYINASLIKMEEAQRSYILTQ…. Peptide  Substring of the protein. LPKNKNRNRYRDVSPFDHSR Trypsin  Aggressive and stable protease  It cleaves proteins very specifically on the carboxy-terminal side (right side) of arginine(R) and lysine(K) residues.

10. Hanyang Univ. Miscleavage(Faulty cleavage) Semitryptic Nontryptic Sample - Protein Digestion

11. Hanyang Univ. Proteomics 11

12. Hanyang Univ. Separation - GEL Separation by 2DE gels Basic scheme 1.separation by pI and mw 2.stain gel (coomassie, silver) 3.semi-quantitation based on spot size/intensity Advantages 1. great for visualizing whole-cell proteome 2. useful for comparative proteomics Disadvantages 1. low expression protein often not visible 2. cannot be automated 12

13. Hanyang Univ. Separation – LC Liquid chromatography is a separation technique that involves:  injection of a small volume of liquid sample  into a tube packed with porous particles (stationary phase)  where individual components of the same sample are transported along the packed column by a liquid moved by gravity. The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between the molecules and the packing particles.

14. Hanyang Univ. Separation - HPLC High-Performance LC is a separation technique that involves:  injection of a small volume of liquid sample  into a tube packed with tiny particles (3-5 micron in diameter called stationary phase)  where individual components of the same sample are moved down the packed column with a liquid (mobile phase) forced through the column by high pressure delivered by pump. The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between the molecules and the packing particles.

15. Hanyang Univ. Proteomics 15

16. Hanyang Univ. Mass spectrometry 16

17. Hanyang Univ. 17 Mass spectrometry 2002 Nobel Prize MALDI matrix-assisted laser desorption ESI electrospray ionization Koichi Danaka John Fenn Soft ionization

18. Hanyang Univ. Mass Spectrometry Ion selection Quadrupole 18 https://www.youtube.com/watch?v=qxPb9vFWdqo

19. Hanyang Univ. Mass Spectrometry TOF Mass analyzer ion selection Ion trap Quadrupole ion detection Time-of-flight (TOF) Fourier-Transform Ion Cyclotron Resonance MS (FT-ICR MS) Common: masses separated by m/z a.ion source b.reflectron c.ion path d.detector 19

20. Hanyang Univ. Mass Spectrometry TOF Mass analyzer m/z can be inferred from time spent in flight. The same force will accelerate each ion differently based on its mass (m) and charge (z). ++ + + + Detector 20

21. Hanyang Univ. Mass Spectrometry 21

22. Hanyang Univ. Single stage MS 22

23. Hanyang Univ. Tandem MS 23

24. Hanyang Univ. MS vs. MS/MS 24

25. Hanyang Univ. Mass vs. Intensity vs. Time 25

26. Hanyang Univ. The mass-spectrometry/proteomic experiment AAAA EEEE CCCC DDDD FFFF KKKK LLLL MMMM AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD GGGG FFFF HHHH KKKK PPPP LLLL MMMM QQQQ TTTT RRRR SSSS IIII PPPP LLLL MMMM QQQQ TTTT RRRR SSSS FFFF IIII GGGG HHHH IIIIGGGGHHHHIIIIPPPPMMMM IIII GGGG HHHH IIII PPPP MMMM FFFF HHHH KKKK PPPP LLLL MMMM QQQQ TTTT RRRR SSSS AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH FFFF HHHH KKKK PPPP MMMM QQQQ TTTT SSSS EEEE CCCC IIII HHHH AAAA HHHH m/z intensity AAAA m/z intensity MMMM m/z intensity KKKK digestion mass-spectrometry Protein mixture Peptide mixture Mass spectra m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity CCCC DDDD PPPP QQQQ LLLL SSSS 26

27. Hanyang Univ. The mass-spectrometry/proteomic experiment Experimental mass spectra m/z intensity m/z intensity m/z intensity Database search De novo sequencing m/z intensity AAAA m/z intensity MMMM m/z intensity KKKK Peptide-spectrum match Peptide assembly Protein assignment AAAA EEEE CCCC DDDD FFFF KKKK LLLL MMMM AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD GGGG FFFF HHHH KKKK PPPP LLLL MMMM QQQQ TTTT RRRR SSSS IIII PPPP LLLL MMMM QQQQ TTTT RRRR SSSS FFFF IIII GGGG HHHH IIIIGGGGHHHHIIIIPPPPMMMM IIII GGGG HHHH IIII PPPP MMMM Peptide assignment Peptide validation FFFF HHHH KKKK PPPP LLLL MMMM QQQQ TTTT RRRR SSSS AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH FFFF HHHH KKKK PPPP MMMM QQQQ TTTT SSSS EEEE CCCC IIII HHHH AAAA HHHH 27

28. Hanyang Univ. Pipeline : integrated tools for MS/MS proteomics Input Spectrum data (Protein database) Peptide assignment SEQUEST PEAKS MODi Peptide validation manual validation PeptideProphet Target/Decoy Protein assignment & validation ProteinProphet IDPicker Output Interpretation Quantitation ASAPRatio MaxQuant 28

29. Hanyang Univ. Peptide assignment >Protein A MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFD HSRKLHQEDNDYINASLIKMEEAQRSYILTQQIDKSGSWAAIYQDIRHEASDF HEASDFPCRVAKLPKNKNRNRYMEKEFEQIDKSGSWAAIYQDIRHEMEKEFEQ IDKSGSWAAIYQDIRHE >Protein B MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQ QQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPE VMGVSKVKEAMAPKHKEMPFPKYPVEPF >Protein C... m/z intensity VAKLPKNKNR 29

30. Hanyang Univ. Peptide validation m/z intensity AAAA m/z intensity m/z intensity CCCC m/z intensity m/z intensity m/z intensity GGGG KKKKLLLL TTTT Peptide-spectrum match m/z intensity m/z intensity m/z intensity LLLLQQQQIIII 30

31. Hanyang Univ. Protein Assignment IIII PPPP LLLLMMMMQQQQ AAAA EEEE CCCC DDDD FFFF KKKK LLLL MMMM AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD FFFF IIII GGGG HHHH AAAA EEEE CCCC DDDD GGGG FFFF HHHH KKKK PPPP LLLL MMMM QQQQ TTTT RRRR SSSS QQQQ TTTT RRRR SSSS FFFF IIII GGGG HHHH IIIIGGGGHHHHIIIIPPPPMMMM IIII GGGG HHHH IIII PPPP MMMM PPPP LLLL QQQQ IIII MMMM FFFF HHHH KKKK MMMM TTTT RRRR SSSS AAAA EEEE CCCC DDDD FFFF GGGG HHHH FFFF HHHH KKKK PPPP QQQQ TTTT SSSS EEEE CCCC IIII HHHH AAAA HHHH Proteins Peptides Mass spectra Missing values m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity LLLLQQQQIIIIPPPPMMMM 31

32. Hanyang Univ. Protein Assignment 32