Mass spectrometric analysis of integral membrane proteins at the subnanomolar level: Application to recombinant photopigments

Integral membrane proteins produced by eukaryotic expression systems are a subject of much current interest in biomedical investigation. Due to the lo w efficiency of their expression and the limited quantity of the expressed to the total amount of the membrane proteins, they have evaded mass spectro metric analysis. The methodology previously developed for mass spectrometri c analysis of integral membrane proteins required proteins that were obtain ed relatively pure from their native membranes. The previously developed me thodology has been modified and applied to the analysis of subnanomolar sam ples of rhodopsin. Bovine rhodopsin purified by affinity chromatography, fr om native membranes and from a eukaryotic expression system, was successful ly analyzed, obtaining complete sequence coverage for the detection and loc alization of posttranslational modifications. The methodology presented her e will enable mass spectrometric analysis of subnanomolar levels of photopi gments or, other integral membrane proteins either from their native membra nes or as products of expression systems.