Lecture24 - Lecture 24: Protein Identification by Mass...

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Lecture 24: Protein Identification by Mass Spectrometry § Identifying proteins and peptides by Mass Spectrometry • Peptide mass fingerprint • MS/MS based peptide ID § MS/MS methodology • Quadrupole Mass Analyzer • MS/MS based peptide ID methodology Adapted from slides originally created by Dr. James Bruce MS Information Analysis for Proteomics 2 Strategies for MS-based protein ID 1) Peptide mass fingerprint 2) MS/MS-based peptide ID Both approaches are carried out with peptides generated by chemical or enzymatic cleavage of proteins Peptide mass fingerprint Cleave protein into a set of peptides Measure peptide masses Search protein database with: (1) measured peptide masses (2) Protein mass, if known (3) known cleavage rules Cleaves before D Asp-N Cleaves after F or L or M or W or Y if not followed by P Chymotrypsin Cleaves after E Glu-C Cleaves after K Lys-C Cleaves after K or R if not followed by P Trypsin Cleaves after M CNBr (Cyanogen Bromide) Cleaves after R Arg-C Sequence Specificity Enzyme (or reagent) Protein Cleavage Rules for Common Proteases
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Average Numbers of Peptide Fragments 38 8 CNBr 9 35 Trypsin 16 19 Lys-C 15 20 Glu-C 6 54 Chymotrypsin 18 17 Asp-N 18 17 Arg-C Average length of peptide fragments # of peptide fragments Enzyme § Expected number of peptide fragments upon cleavage of a 300 amino acid protein: Data from Kellner et al. (1994) Trypsin digestion is the most commonly used cleavage method Larger size peptides more unique mass (mass fingerprint)
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This note was uploaded on 01/20/2012 for the course MBIOS 478 taught by Professor Staff during the Fall '11 term at Washington State University .

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Lecture24 - Lecture 24: Protein Identification by Mass...

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