title: Evaluation of enzymatic digestion and liquid chromatography-mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin.
abstract: A method for the complete peptide mapping of the model integral membrane protein bacteri-orhodopsin is demonstrated. Utilizing more effective enzymatic digestion, procedures with capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (MS/MS), all predicted tryptic digestion products were detected, as well as peptides from all previously reported post-translational modifications of bacteriorhodopsin. A significant contribution of chymotryptic-like digestion products was also observed. A characterization of the behavior of hydrophobic integral membrane peptides in a reversed-phase liquid chromatographic separation is also provided. The method reported here offers improved compatibility of the solubilizing reagents with both the chromatography and mass spectrometry, rendering it suitable for high-throughput proteomic applications.
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Kim K
authors.initials: KK
authors.lastname: Hixson
authors.forename: Nestor
authors.initials: N
authors.lastname: Rodriguez
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Eric F
authors.initials: EF
authors.lastname: Strittmatter
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D010449:Peptide Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D001436:Bacteriorhodopsins; D014357:Trypsin