title: Tryptic digestion of in-gel proteins for mass spectrometry analysis.
abstract: Identification and characterization of proteins are ultimately the goal in proteomic analysis. In order to identify a protein trypsin is commonly used to digest protein into peptides which can be analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). This chapter describes a tryptic digestion method for digestion of proteins in one-dimensional (1DE) or two-dimensional (2DE) polyacrylamide gels. The method involves cutting target protein bands or spots, removal of protein stain, reduction and alkylation of native protein, digestion and finally extraction of peptides for mass spectrometry analysis. The method is simple and reasonably sensitive that many in-gel proteins that are barely visible with Coomassie blue stain have been successfully identified.
authors.affiliation: Minomic Pty LtdLevel 3, 126 Greville St, Chatswood West, NSW, 2067, Australia.
authors.forename: Mai-Loan
authors.initials: ML
authors.lastname: Huynh
authors.forename: Pamela
authors.initials: P
authors.lastname: Russell
authors.forename: Bradley
authors.initials: B
authors.lastname: Walsh
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D011506:Proteins; D014357:Trypsin