Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS.
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).
Choudhary
Gargi
G
Thermo Finnigan, San Jose, California 95134, USA.
Wu
Shiaw-Lin
SL
Shieh
Paul
P
Hancock
William S
WS
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
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