Mass spectrometric analysis of integral membrane proteins at the subpicomolar level: application to rhodopsin.
Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.
Ablonczy
Zsolt
Z
Department of Ophthalmology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
Crouch
Rosalie K
RK
Knapp
Daniel R
DR
D000818:Animals; D002417:Cattle; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D036103:Nanotechnology; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping / Q000379:methods; D012243:Rhodopsin / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity
D008565:Membrane Proteins; D010446:Peptide Fragments; D012243:Rhodopsin; D003545:Cysteine; D003488:Cyanogen Bromide