1. Lecture-9 MS Techniques and Protein Identification Huseyin Tombuloglu, Phd GBE423 Genomics & Proteomics

2. Diversity of Proteins Separating Proteins 2D gel electrophoresis, mass spectrometry Identifying Proteins peptide fingerprinting, de novo sequencing Quantifying Proteins DIGE, ICAT Protein interactions high-throughput mass spectrometry

3. Techniques for the analysis of proteins and proteomes Rainer Breitling Groningen Bioinformatics Centre

4. Diversity of proteins Hydrophobicity Polarity Acidity Charge Size Proteins are much more diverse

5. Diversity of proteins More than 5% of all genes can produce variant proteins by alternative splicing.

6. Diversity of proteins Protein targeting and trafficking: to nucleus or mitochondria to vesicular apparatus to peroxisomes to membranes and extracellular space

7. Diversity of proteins phosphorylation, the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine  kinase signalling cascades acetylation, the addition of an acetyl group, usually at the N-terminus of the protein alkylation, the addition of an alkyl group (e.g. methyl, ethyl) methylation the addition of a methyl group, usually at lysine or arginine residues (this is a subtype of alkylation)  histone modification isoprenylation, the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol)  membrane targeting glycosylation, the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein  extracellular stability Tyrosine sulfation, the addition of a sulfate group to a tyrosine. ubiquitination, the covalent linkage of the protein ubiquitin to a target protein  protein degradation/trafficking SUMOylation, the covalent linkage of the SUMO protein (Small Ubiquitin-related MOdifier) to a target protein.

8. Conventional Proteomic Approach Excise gel spot Tryptic digest 2D-PAGE Destain Proteins from lysed cell Reduce w/DTT Alkylate w/IAA OR Matrix Assisted Laser Desorption Ionization - Mass Spectrometry Electrospray Ionization – Mass Spectrometry

9. 2D gel electrophoresis isoelectric focusing  separation by pI SDS gel electrophoresis  separation by mass

10. 2D gel electrophoresis Control Experimental

11. 2D gel electrophoresis Control Experimental PTM? Downregulation?

16. Mass Spectrometer Ionizer Sample + _ Mass Analyzer Detector MALDI Electro-Spray Ionization (ESI) Time-Of-Flight (TOF) Quadrupole Ion-Trap Electron Multiplier (EM)

17. MALDI-TOF-MS

18. Mass Spectrometer (MALDI-TOF) Source Length = s Field-free drift zone Length = D E d = 0 Microchannel plate detector Backing plate (grounded) Extraction grid (source voltage -V s ) UV (337 nm) Detector grid -V s Pulse voltage Analyte/ matrix

19. Electrospray Ionization-MS Quadrupole time-of-flight (Q-TOF) CID spectra (collision induced dissociation) are obtained from the MS/MS mass analyzer. Ion Source Mass Analyzer Detector (2) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207.

20. Peptide Mass Fingerprint Trypsin Digest This section is taken from Nathan Edwards, UMIACS

21. Peptide Mass Fingerprint MS

22. Peptide Mass Fingerprint Trypsin: digestion enzyme –Highly specific –Cuts after K & R except if followed by P Protein sequence from sequence database –In silico digest –Mass computation For each protein sequence in turn: –Compare computer generated masses with observed spectrum

23. Protein Sequence Myoglobin - Plains zebra GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG

24. Protein Sequence Myoglobin - Plains zebra GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG

25. Peptide Masses 1811.90 GLSDGEWQQVLNVWGK 1606.85 VEADIAGHGQEVLIR 1271.66 LFTGHPETLEK 1378.83 HGTVVLTALGGILK 1982.05 KGHHEAELKPLAQSHATK 1853.95 GHHEAELKPLAQSHATK 1884.01 YLEFISDAIIHVLHSK 1502.66 HPGDFGADAQGAMTK 748.43 ALELFR

26. Peptide Mass Fingerprint GLSDGEWQQVLNVWGK VEADIAGHGQEVLIR LFTGHPETLEK HGTVVLTALGGILK KGHHEAELKPLAQSHATK GHHEAELKPLAQSHATK YLEFISDAIIHVLHSK HPGDFGADAQGAMTK ALELFR

27. Mass Spectrometry Strengths –Precise molecular weight –Fragmentation –Automated Weaknesses –Best for a few molecules at a time –Best for small molecules –Mass-to-charge ratio, not mass –Intensity ≠ Abundance

28. Single Stage MS MS

29. Tandem Mass Spectrometry (MS/MS) Precursor selection

30. Tandem Mass Spectrometry (MS/MS) Precursor selection + collision induced dissociation (CID) MS/MS

31. Peptide Fragmentation H…-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH R i-1 RiRi R i+1 AA residue i-1 AA residue i AA residue i+1 N-terminus C-terminus Peptides consist of amino-acids arranged in a linear backbone.

32. i+1 Peptide Fragmentation -HN-CH-CO-NH-CH-CO-NH- RiRi CH-R’ bibi y n-i y n-i-1 b i+1 R” i+1

33. Peptide Fragmentation Peptide: S-G-F-L-E-E-D-E-L-K MWion MW 88b1b1 S GFLEEDELKy9y9 1080 145b2b2 SG FLEEDELKy8y8 1022 292b3b3 SGF LEEDELKy7y7 875 405b4b4 SGFL EEDELKy6y6 762 534b5b5 SGFLE EDELKy5y5 633 663b6b6 SGFLEE DELKy4y4 504 778b7b7 SGFLEED ELKy3y3 389 907b8b8 SGFLEEDE LKy2y2 260 1020b9b9 SGFLEEDEL Ky1y1 147

34. Peptide Fragmentation 100 0 2505007501000 m/z % Intensity K 1166 L 1020 E 907 D 778 E 663 E 534 L 405 F 292 G 145 S 88b ions 147260389504633762875102210801166y ions

35. Peptide Fragmentation K 1166 L 1020 E 907 D 778 E 663 E 534 L 405 F 292 G 145 S 88b ions 100 0 2505007501000 m/z % Intensity 147260389504633762875102210801166y ions y6y6 y7y7 y2y2 y3y3 y4y4 y5y5 y8y8 y9y9 b3b3 b5b5 b6b6 b7b7 b8b8 b9b9 b4b4

36. Peptide Identification Given: The mass of the precursor ion, and The MS/MS spectrum Output: The amino-acid sequence of the peptide

37. Sequence Database Search Sequence fills in gaps in the spectrum All candidates have biological relevance Practical for high-throughput peptide identification Correct peptide might be missing from database!

38. Mascot Search Engine

39. Mascot MS/MS Ions Search

40. Mascot MS/MS Search Results