Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.
Barnidge
D R
DR
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717-3400, USA.
Dratz
E A
EA
Sunner
J
J
Jesaitis
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
D008565:Membrane Proteins; D010446:Peptide Fragments; D012243:Rhodopsin; D014357:Trypsin