Protein Analysis of Human Sperm

Sperm are highly specialized cells, with 1,056 and 1,760 proteins identified in the sperm proteome according to previous studies.^1,² As members of the Chromosome-Centric Human Proteome Project,³ the Xuejiang Guo and Jiahao Sha groups used advanced proteomic techniques to identify nearly four times the number of proteins identified previously.

In a published study from these groups,⁴ sperm samples were obtained from 32 healthy male volunteers with normal sperm production. Sperm were purified and washed to remove seminal plasma, germ-line, and other non-sperm cells. The samples were pooled together and analyzed using capillary reverse-phase liquid chromatography and mass spectrometry (LC-MS/MS) using a LTQ Orbitrap Velos mass spectrometer (Thermo Scientific).

Raw files from LC-MS/MS were loaded into protein databases for analysis. A false discovery rate (FDR) criteria of 1% was developed by searching a database with the reversed amino acid sequence.⁵ To be counted in the total number of proteins identified, the proteins needed to contain a minimum of six amino acids and also possess an FDR of 1%. 30,903 unique peptides were discovered, corresponding to 4,675 proteins. Of those proteins, 4,401 of those proteins were identified again in two repeat experiments.

Comparisons with published sperm nucleus proteomes further validated the proteins identified. 206/221 proteins (93%) were successfully identified when compared with two independent studies, while 3,777 new proteins were identified in this study.

Immunofluorescence studies of 29 randomly selected proteins further validated protein identification by illustrating their presences in a variety of sperm structures. Functional pathways in human sperm also included overexpression of the IL-6 insulin and TGF-beta pathways.

Known sperm drug targets were identified using the DrugBank database. 500 proteins had known drug targets. 154 were determined to be mitochondrial proteins responsible for giving sperm the energy needed for motility, and 162 proteins were cilia proteins, which aid in movement. Further understanding of these drug targets may lead to the development of male factor infertility treatments and male contraceptives.

References

1. Baker, M.A., et al. (2007) ‘Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis‘, Proteomics Clinical Applications, 1 (5), (pp. 524-532)

2. Johnston, D.S., et al. (2005) ‘Analysis of the human sperm proteome‘, Annals of the New York Academy of Sciences, Dec 1061 , (pp. 190-202)

3. Paik, Y.K., et al., (2012) ‘The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome‘, Nature Biotechnology, 30 (pp. 221-223)

4. Wang, G., et al. (2013) ‘In-depth proteomic analysis of the human sperm reveals complex protein compositions‘, Journal of Proteomics, 79, (pp. 114-122)

5. Cox, J., et al. (2011) ‘Andromeda: a peptide search engine integrated into the MaxQuant environment‘, Journal of Proteome Research, 10 (4), (pp. 1794-1805)