High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins

In higher plants, both photosystem I (PSI) and II (PSII) consist of membran e-embedded proteins that contain more than one transmembrane alpha helix. P SI is a multiprotein complex consisting of a core complex of thirteen prote ins surrounded by four different types of light harvesting antenna proteins . Up to now, the protein components of both photosystems have been characte rized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not suff icient to discriminate between individual membrane proteins. This is also c omplicated by the fact that some proteins, such as the antenna proteins, ha ve almost identical molecular mass and amino acid sequence, making it diffi cult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins an d most of the core components of PSI from spinach, together with the identi fication of proteins by molecular mass, successfully deduced by the combine d use of HPLC coupled on-line with a mass spectrometer equipped with an ele ctrospray ion source (ESI-MS), The proposed RP-HPLC-ESI-MS method holds sev eral advantages over SDS-PAGE, including better protein separation, especia lly for antenna proteins, mass accuracy, speed, efficiency, and the potenti al to reveal isomeric forms. Moreover, the molecular masses determined by H PLC-ESI-MS are in good agreement with the molecular masses of the individua l components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of posttranslational modifications in the se proteins. It follows that if the method proposed is useful for these hig hly hydrophobic proteins, it may be of general use for any membrane protein s, where the presence of detergent for solubilization may compromise their characterization. (C) 2000 Wiley-Liss, Inc.