Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal more protein spots, immobilized pH gradient strips （24 cm） of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient （IPG） strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results： The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip （1 306 spots）. Conclusion： The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.
Ling-Wei Li Li-Qing Fan Wen-Bing Zhu Hong-Chuan Nie Bo-Lan Sun Ke-Li Luo Ting-Ting Liao Le Tang Guang-Xiu Lu
