title: Fast-response proteomics by accelerated in-gel digestion of proteins.
abstract: Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Havlis
authors.forename: Henrik
authors.initials: H
authors.lastname: Thomas
authors.forename: Marek
authors.initials: M
authors.lastname: Sebela
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D004718:Saccharomycetales / Q000737:chemistry; D012568:Schizosaccharomyces / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011506:Proteins; D014357:Trypsin