The ARC Centre of Excellence in Biotechnology and Development, Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia.
J Proteome Res. 2010 Feb 5;9(2):718-29. doi: 10.1021/pr900513d.
Before fertilization can occur, ejaculated mammalian spermatozoa must undergo a maturation process known as capacitation, which is dominated by post-translational modifications, particularly phosphorylation. Despite its biological importance, characterization of those proteins targeted for phosphorylation during capacitation remains ill-defined. Here, we report the isolation and purification of 288 phosphorylated peptides from rat spermatozoa using titanium dioxide columns in combination with nanoflow mass spectrometry. This equated to 120 identified phosphorylated proteins present in pure populations of spermatozoa. The MS survey scans of replicate titanium dioxide eluates, derived from both noncapacitated and capacitated sperm lysates, were then compared in silico using a virtual 2D PAGE format and DeCyderMS software. This analysis found 15 differentially phosphorylated proteins during capacitation. Included in this list were sperm qualifiers such as Izumo, a known sperm-oocyte fusion protein. To demonstrate that this label-free quantitative approach to phosphoprotein analysis was viable, we measured the enzymatic activity of 5'-nucleotidase, the phosphorylation status of which changed during capacitation. The results revealed, for the first time, that 5'-nucleotidase activity is up-regulated as sperm capacitate. This change, together with the other protein identifications reported in this study, constitute important new leads in elucidating the biochemical mechanisms by which spermatozoa attain a capacitated state.
在受精发生之前，射出的哺乳动物精子必须经历一个成熟过程，称为获能，其主要由翻译后修饰，特别是磷酸化来主导。尽管它具有生物学重要性，但在获能过程中针对磷酸化的那些蛋白质的特征仍然不清楚。在这里，我们报告了使用二氧化钛柱结合纳流质谱从大鼠精子中分离和纯化 288 个磷酸化肽。这相当于在纯精子群体中存在 120 个鉴定出的磷酸化蛋白质。然后，使用虚拟 2D PAGE 格式和 DeCyderMS 软件，对来自未获能和获能精子裂解物的重复二氧化钛洗脱物的 MS 扫描进行了计算机比较。该分析发现，在获能过程中有 15 个磷酸化蛋白差异表达。其中包括 Izumo 等精子鉴定因子，Izumo 是一种已知的精子-卵母细胞融合蛋白。为了证明这种无标记定量磷酸蛋白分析方法是可行的，我们测量了 5'-核苷酸酶的酶活性，其磷酸化状态在获能过程中发生变化。结果首次揭示，5'-核苷酸酶活性在精子获能时上调。这种变化以及本研究中报告的其他蛋白质鉴定构成了阐明精子获得获能状态的生化机制的重要新线索。
