A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X-100 soluble and insoluble proteins were recovered and separated by SDS-PAGE. The separation lanes were dissected into 96 fractions and analyzed individually by LC-MS(n) . A comprehensive protocol, involving LC-MS/MS analysis eventually down to the ninth most intense peak found in the MS-survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual-oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven-pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate-gated ion channel receptors, transient receptor potential cation channels, and a non-genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC-MS/MS analysis.
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