Identification of glyceraldehyde-3-phosphate dehydrogenase by protein sequencing in the rat postsynaptic density...

Although many abundant proteins of the postsynaptic density (PSD) are known, most of the less abundant and minor PSD proteins await identification. In this work we attempted to identify a 37 kDa protein, which represented less than 1% of the total n-octyl glucoside (NOG)-insoluble proteins, by protein sequencing. To enrich the target protein, the NOG-insoluble fraction was first electrophoresed in 6% SDS-polyacrylamide gels, and the proteins smaller than 45 kDa compressed in the gel from were electroeluted and subsequently reseparated in 10% SDS-gels. This procedure enriched the target protein to represent about 25% of the eluted proteins. Peptides were generated by digesting the target protein with trypsin directly in the gel and purified by a reverse phase high performance liquid chromatography (HPLC). Two peptides were determined for amino acid sequences. A database search revealed that both sequence were found in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with a minor discrepancy, indicating the the 37 kDa protein in the NOG-insoluble pSD fraction is an isoform of GAPDH.