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

This review of current HPLC techniques concludes that they represent a valuable tool for the characterization of virtually any hydrophobic protein, given the wide versatility, relative ease of use, and high resolution of the reversed phase column. Moreover, since the procedure does not destroy the sample, it allows for protein identification by coupling the column outlet on line with a mass spectrometer interfaced with an electrospray source. Thus, using the thylakoid membrane of the photosynthetic apparatus as a model, we have demonstrated that by taking intact mass measurements (IMM), each protein may be identified on the basis of the close correspondence between the molecular masses measured by RP-HPLC-ESI-MS with those expected from the DNA sequence, in those cases where post-translational modifications may be supposed absent. This was corroborated by the evidence that proteins assigned by IMM are also confirmed by 'in solution' trypsin digestion and peptide fragment fingerprinting (PFF) of each protein isolated by RP-HPLC using a preparative scale column. On the other hand, even when denaturated these highly hydrophobic proteins are barely digested, resulting the small number of peptides not sufficient for unequivocal protein identification by mass peptide fingerprinting. Furthermore, because IMM reflects the full protein sequence, it is possible to elucidate in a short time whether the protein has undergone any post-translational modifications. In the presence of single or multiple phosphorylations, for example, it is possible to estimate approximately the amount of phosphorylated protein present as a percentage of the total protein by comparing the intensity of deconvolution of each protein.