UMR INRA-CNRS, Nouzilly, France.
Proteomics. 2011 May;11(10):1952-64. doi: 10.1002/pmic.201000662. Epub 2011 Apr 7.
Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.
精子表面膜蛋白在精子与卵子的连续相互作用中起着关键作用。本研究旨在使用一种新的整体方法研究精子表面膜蛋白，以确定精子发生后分化过程中发生的蛋白变化。为此目的，开发了一种基于用磺基-NHS-SS-生物素标记的表面膜蛋白的特异性纯化的专用方案。然后，使用适当的凝胶电泳分离和纯化方法以及标准蛋白质组学方法，从未成熟和成熟精子中鉴定和定量表面膜蛋白。通过差异溶解将膜相关蛋白与整合膜蛋白区分开来。通过比较整个精子和顶体周围精子质膜的表面蛋白提取物，实现了精子表面蛋白的区域化。鉴定出几种已知蛋白和与附睾成熟过程相关的新蛋白，表明该方案可特异性纯化亚蛋白组并鉴定新的精子膜蛋白，具有可靠性。这种方法为寻找男性生育标志物开辟了一个新的领域。
