title: Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems.
abstract: Mass spectrometry-based identification of the components of multiprotein complexes often involves solution-phase proteolytic digestion of the complex. The affinity purification of individual protein complexes often yields nanogram to low-microgram amounts of protein, which poses several challenges for enzymatic digestion and protein identification. We tested different solvent systems to optimize trypsin digestions of samples containing limited amounts of protein for subsequent analysis by LC-MS-MS. Data collected from digestion of 10-, 2-, 1-, and 0.2-microg portions of a protein standard mixture indicated that an organic-aqueous solvent system containing 80% acetonitrile consistently provided the most complete digestion, producing more peptide identifications than the other solvent systems tested. For example, a 1-h digestion in 80% acetonitrile yielded over 52% more peptides than the overnight digestion of 1 microg of a protein mixture in purely aqueous buffer. This trend was also observed for peptides from digested ribosomal proteins isolated from Rhodopseudomonas palustris. In addition to improved digestion efficiency, the shorter digestion times possible with the organic solvent also improved trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent. The technique was also demonstrated for an affinity-isolated protein complex, GroEL. To our knowledge, this report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity.
authors.affiliation: Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
authors.forename: Michael Brad
authors.initials: MB
authors.lastname: Strader
authors.forename: David L
authors.initials: DL
authors.lastname: Tabb
authors.forename: W Judson
authors.initials: WJ
authors.lastname: Hervey
authors.forename: Chongle
authors.initials: C
authors.lastname: Pan
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Hurst
mesh_terms: D000097:Acetonitriles; D018834:Chaperonin 60 / Q000378:metabolism; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D012270:Ribosomes / Q000378:metabolism; D031804:Rumex / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology*; D014867:Water
chemical_list: D000097:Acetonitriles; D018834:Chaperonin 60; D010446:Peptide Fragments; D011506:Proteins; D014867:Water; D014357:Trypsin; C032159:acetonitrile