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 low efficiency of their expression and the limited quantity of the expressed to the total amount of the membrane proteins, they have evaded mass spectrometric analysis. The methodology previously developed for mass spectrometric analysis of integral membrane proteins required proteins that were obtained relatively pure from their native membranes. The previously developed methodology has been modified and applied to the analysis of subnanomolar samples of rhodopsin. Bovine rhodopsin purified by affinity chromatography, from native membranes and from a eukaryotic expression system, was successfully analyzed, obtaining complete sequence coverage for the detection and localization of posttranslational modifications. The methodology presented here will enable mass spectrometric analysis of subnanomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.
Ablonczy
Z
Z
Department of Ophthalmology, Medical University of South Carolina, Charleston 29425, USA.
Kono
M
M
Crouch
R K
RK
Knapp
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D003488:Cyanogen Bromide / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis; D012243:Rhodopsin / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012243:Rhodopsin; D003488:Cyanogen Bromide