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Evaluation of enzymatic digestion and liquid chromatography‐mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin
Author(s) -
Hixson Kim K.,
Rodriguez Nestor,
Camp David G.,
Strittmatter Eric F.,
Lipton Mary S.,
Smith Richard D.
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200209)23:18<3224::aid-elps3224>3.0.co;2-#
Subject(s) - bacteriorhodopsin , chromatography , mass spectrometry , chemistry , integral membrane protein , peptide mapping , peptide , digestion (alchemy) , liquid chromatography–mass spectrometry , protein mass spectrometry , membrane protein , membrane , biochemistry , tandem mass spectrometry , peptide sequence , gene
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.