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Introduction to Mass Spectrometry-based Proteomics Hanyang Univ. 1 Central dogma of molecular biology DNA mRNA transcription Protein translation Hanyang Univ. 2 Alternative Splicing exon3 exon2 exon1 exon4 IAMTHEQSRTV FIRST QPBASRTVSECONDQABDECTV ALTERNATIVE intron1 intron2 intron3 IAMTHEFIRSTALTERNATIVE I AM THE FIRST ALTERNATIVE Hanyang Univ. 3 Alternative Splicing exon3 exon2 exon1 exon4 IAMTHEQSRTV FIRST QPBASRTVSECONDQABDECTV ALTERNATIVE intron1 intron2 intron3 IAMTHESECONDALTERNATIVE I AM THE SECOND ALTERNATIVE Hanyang Univ. 4 Proteomics Post-translational Modification (PTM) conformational change change in cellular localization protein-protein interaction Various Cellular Functions Hanyang Univ. 5 Genomics vs. Proteomics Blueprint vs. Machine DNA Proteins Easily amplified by PCR No 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 Hanyang Univ. 6 Proteomics: study of protein Protein expression Proteinprotein interaction Protein structure Hanyang Univ. 7 Proteomics http://www.childrenshospital.org/research/_proteomics /index.html Hanyang Univ. 8 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. Hanyang Univ. Sample - Protein Digestion Miscleavage(Faulty cleavage) Semitryptic Nontryptic Hanyang Univ. Proteomics Hanyang Univ. 11 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 Hanyang Univ. 12 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. 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. Hanyang Univ. Proteomics Hanyang Univ. 15 Mass spectrometry Hanyang Univ. 16 Mass spectrometry 2002 Nobel Prize Soft ionization MALDI matrix-assisted laser desorption Koichi Danaka ESI electrospray ionization John Fenn Hanyang Univ. 17 Mass Spectrometry Ion selection Quadrupole https://www.youtube.com/watch?v=qxPb9vFWdqo Hanyang Univ. 18 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. b. c. d. ion source reflectron ion path detector Hanyang Univ. 19 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 + + Hanyang Univ. 20 Mass Spectrometry Hanyang Univ. 21 Single stage MS Hanyang Univ. 22 Tandem MS Hanyang Univ. 23 MS vs. MS/MS Hanyang Univ. 24 Mass vs. Intensity vs. Time Hanyang Univ. 25 The mass-spectrometry/proteomic experiment Peptide mixture HHHH Protein mixture EEEE FFFF KKKK QQQQ HHHH SSSS HHHH AAAA CCCC RRRR TTTT SSSS AAAA LLLL AAAA KKKK CCCC QQQQ EEEE PPPP PPPP HHHH DDDD IIII FFFF GGGG IIII FFFF TTTT MMMM HHHH digestion IIIIPPPPMMMMGGGGHHHHIIII MMMM mass-spectrometry m/z m/z m/z intensity intensity intensity m/z KKKK LLLL SSSS intensity m/z MMMM PPPP QQQQ intensity intensity intensity AAAA CCCC DDDD intensity intensity Mass spectra m/z m/z m/z m/z Hanyang Univ. 26 The mass-spectrometry/proteomic experiment Protein assignment Peptide assignment HHHH KKKK EEEE Peptide assembly IIIIPPPPMMMMGGGGHHHHIIII HHHH FFFF QQQQ HHHH MMMM SSSS HHHH RRRR AAAA CCCC TTTT SSSS AAAA LLLL AAAA KKKK CCCC QQQQ EEEE PPPP PPPP HHHH DDDD IIII FFFF GGGG IIII FFFF TTTT MMMM Peptide validation Experimental mass spectra Peptide-spectrum match KKKK intensity intensity m/z MMMM intensity intensity AAAA intensity intensity Database search De novo sequencing m/z m/z m/z m/z m/z Hanyang Univ. 27 Pipeline : integrated tools for MS/MS proteomics Input Spectrum data (Protein database) Peptide assignment SEQUEST PEAKS MODi Quantitation ASAPRatio MaxQuant Peptide validation manual validation PeptideProphet Target/Decoy Protein assignment & validation ProteinProphet IDPicker Output Interpretation Hanyang Univ. 28 intensity Peptide assignment m/z >Protein A MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFD HSRKLHQEDNDYINASLIKMEEAQRSYILTQQIDKSGSWAAIYQDIRHEASDF HEASDFPCRVAKLPKNKNRNRYMEKEFEQIDKSGSWAAIYQDIRHEMEKEFEQ IDKSGSWAAIYQDIRHE >Protein B MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQ QQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPE VMGVSKVKEAMAPKHKEMPFPKYPVEPF >Protein C ... VAKLPKNKNR ... Hanyang Univ. 29 Peptide validation Peptide-spectrum match CCCC GGGG intensity intensity intensity AAAA m/z m/z m/z LLLL TTTT intensity intensity intensity KKKK m/z m/z m/z intensity IIII intensity QQQQ intensity LLLL m/z m/z m/z Hanyang Univ. 30 Protein Assignment Peptides Proteins HHHH EEEE KKKK FFFF QQQQ HHHH SSSS HHHH AAAA CCCC RRRR TTTT SSSS AAAA LLLL AAAA KKKK CCCC QQQQ EEEE PPPP PPPP HHHH DDDD IIII FFFF GGGG IIII FFFF TTTT MMMM HHHH IIIIPPPPMMMMGGGGHHHHIIII MMMM Missing values Mass spectra m/z m/z MMMM m/z intensity intensity PPPP intensity IIII intensity QQQQ intensity LLLL m/z m/z Hanyang Univ. 31 Protein Assignment Hanyang Univ. 32