Shetty J, Diekman A B, Jayes F C, Sherman N E, Naaby-Hansen S, Flickinger C J, Herr J C
Department of Cell Biology, University of Virginia, Charlottesville 22908-0732, USA.
Electrophoresis. 2001 Aug;22(14):3053-66. doi: 10.1002/1522-2683(200108)22:14<3053::AID-ELPS3053>3.0.CO;2-K.
The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.
本研究的目的是发现先前未知的人类精子表面蛋白，这些蛋白可能是候选避孕疫苗抗原。为此，采用了通过质谱对人类精子蛋白进行浓缩以用于微测序的方法，这增加了鉴定表面蛋白的可能性。采用向量标记、差异提取和二维（2-D）凝胶电泳来鉴定和分离细胞表面可及的蛋白。对Percoll梯度离心收集的精子或上游法获得的精子，要么直接进行溶解，要么在用磺基琥珀酰亚胺基-6-（生物素酰胺基）己酸酯（磺基-NHS-LC-生物素）进行表面标记后再进行溶解。对用四种裂解缓冲液提取的蛋白进行了比较：（i）含有9.8 M尿素和2%聚乙二醇辛基苯基醚（Igepal CA-630）的Celis缓冲液；（ii）1% Triton X（TX）-100；（iii）1.7% TX-114，随后进行相分离；或（iv）1 M NaCl。用抗生物素蛋白以及针对三个结构域的已知蛋白的抗体对通过高分辨率二维电泳分离的蛋白印迹进行检测，这三个结构域分别是：精子表面（SAGA-1）、顶体（SP-10）和细胞骨架（α-微管蛋白）。Celis缓冲液（45分钟）从所有三个主要区室中提取蛋白。然而，在Celis缓冲液中进行20秒的提取可富集多种蛋白，并能够通过质谱鉴定出几种新的肽段。用TX-100或1 M NaCl进行温和提取主要溶解了膜蛋白和顶体蛋白，但未溶解细胞骨架蛋白。对每种方法提取的生物素化蛋白进行比较表明，Celis缓冲液溶解的主要向量标记蛋白也可被TX-100、TX-114和1 M NaCl溶解。用TX-114提取后进行相分离可显著富集疏水表面蛋白，并有助于分辨率和分离。在所有这些提取方法之后进行微测序的八个蛋白斑点被证明是新的精子分子。
