Liquid Extraction-Ultracentrifugation-Liquid Chromatography-Mass Spectrometry: A Potent Tool for Separation and Identification of Thylakoid Membrane Proteins

摘要

This review of current HPLC techniques concludes that they represent a valuable tool for thencharacterization of virtually any hydrophobic protein, given the wide versatility, relative ease of use, and highnresolution of the reversed phase column. Moreover, since the procedure does not destroy the sample, it allowsnfor protein identification by coupling the column outlet on line with a mass spectrometer interfaced with annelectrospray source. Thus, using the thylakoid membrane of the photosynthetic apparatus as a model, we havendemonstrated that by taking intact mass measurements (IMM), each protein may be identified on the basis ofnthe close correspondence between the molecular masses measured by RP-HPLC-ESI-MS with those expectednfrom the DNA sequence, in those cases where post-translational modifications may be supposed absent. Thisnwas corroborated by the evidence that proteins assigned by IMM are also confirmed by ‘in solution’ trypsinndigestion and peptide fragment fingerprinting (PFF) of each protein isolated by RP-HPLC using a preparativenscale column. On the other hand, even when denaturated these highly hydrophobic proteins are barelyndigested, resulting the small number of peptides not sufficient for unequivocal protein identification by massnpeptide fingerprinting. Furthermore, because IMM reflects the full protein sequence, it is possible to elucidatenin a short time whether the protein has undergone any post-translational modifications. In the presence ofnsingle or multiple phosphorylations, for example, it is possible to estimate approximately the amount ofnphosphorylated protein present as a percentage of the total protein by comparing the intensity ofndeconvolution of each protein.