title: Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS.
abstract: This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).
authors.affiliation: Thermo Finnigan, San Jose, California 95134, USA.
authors.forename: Gargi
authors.initials: G
authors.lastname: Choudhary
authors.forename: Shiaw-Lin
authors.initials: SL
authors.lastname: Wu
authors.forename: Paul
authors.initials: P
authors.lastname: Shieh
authors.forename: William S
authors.initials: WS
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002845:Chromatography; D010450:Endopeptidases; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007477:Ions; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001798:Blood Proteins; D006023:Glycoproteins; D007477:Ions; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010450:Endopeptidases; D014357:Trypsin; D010959:Tissue Plasminogen Activator; D016282:Aspartic Acid Endopeptidases; C044574:lysine-C peptidase; D008239:Lysine