title: Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics.
abstract: Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of Halobacterium salinarum peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5-10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins.
authors.affiliation: Department of Chemistry, University of Texas at Austin, 105 East 24th Street, Austin, Texas 78712, United States.
authors.forename: Sylvester M
authors.initials: SM
authors.lastname: Greer
authors.affiliation: Department of Chemistry, University of Texas at Austin, 105 East 24th Street, Austin, Texas 78712, United States.
authors.forename: W Ryan
authors.initials: WR
authors.lastname: Parker
authors.affiliation: Department of Chemistry, University of Texas at Austin, 105 East 24th Street, Austin, Texas 78712, United States.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D019843:Archaeal Proteins; D010455:Peptides; D010447:Peptide Hydrolases
keywords: GluC; HCD; Halobacterium salinarum; LysC; UVPD; bottom-up proteomics; chymotrypsin; peptides; protease; trypsin