Global gene and protein expression analyses have had great impacts on scientific progresses in this new era of bioinformatics. Although studies using murine and human materials can fully exploit the large volume of their databases, there are quite a few inconveniences for an investigation on non-human primate materials due to still insufficient data collections. Here we examined the availability of human databases for the protein identification process using the two-dimensional electrophoresis-based proteomic study in cynomolgus monkey embryonic stem (ES) cells. Querying public human protein databases, we successfully identified multiple protein spots via mass spectrometric analysis using MALDI-TOF apparatus. The results of the protein identification were confirmed by western blotting using polyclonal antibodies raised against human epitopes. Interestingly, the results of western blotting further identified the existence of previously unreported multiple isoforms of common proteins including glycolytic pathway enzymes. Thus, combined analyses of the mass spectrometry querying the Homo sapience databases and the western blotting using polyclonal antibodies is highly effective in determining protein expressions in monkey cells. Our success in obtaining a draft protein expression profile of cynomolgus monkey ES cells will contribute to the promotion of non-human primate ES cell researches.

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Using a 5-20% (linear gradient) polyacrylamide slab gel electrophoresis, we studied the urinary protein collected from a 14-year-old girl with osteosarcoma during high-dose methotrexate (MTX) and leucovorin rescue therapy. A distinct protein band of approximately 30 kDa (P-30 protein) was observed in addition to albumin. We have never before encountered a 30 kDa urinary protein. Therefore, we first studied the P-30 protein by peptide mass fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We obtained the amino acid sequence “YDPSLKPLSVSYDQATSLR”, which is the same as that of amino acids (40-58) of human carbonic anhydrase II (CAII). Then, we confirmed this protein by Western blotting using anti-human CAII antibody. This enzyme distributes widely in the human bodies, in erythrocytes, kidney, and pancreas. We considered that this CAII originated from the kidney in association with high-dose MTX and leucovorin rescue therapy.

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