Separation and structural analysis of IgG-binding minor proteins in human plasma

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We have examined the origin of human plasma minor proteins which we found comparing the patterns of two-dimensional gel electrophoresis run under two different conditions of size-separation (second dimension electrophoresis) ; one in the absence of denaturing agents throughout the run and one in the presence of SDS during the size-separation electrophoresis. The minor proteins were analyzed by mass spectrometry, identification of proteins by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flignt mass spectrometer (MALDI-TOF MS) and partial amino acid sequencing by mass-tag method using electrospray ionization tandem mass spectrometer (ESI-MS/MS). Further, we have developed a three-step electrophoresis technique which employ slab-agarose gels in the step of isoelectric focusing and showed that this is useful for the structural analysis of the minor proteins. The comparisons of the patterns obtained by using different conditions of two-dimensional electrophoresis, together with the structural analyses of the proteins on the patterns, will provide insights on the construction processes of functional protein complexes.