富集低丰度蛋白的人类精子全蛋白二维电泳整合图的建立

目的:运用二维电泳(2-DE)技术分离人类正常精子低丰度蛋白并建立全精子蛋白的2-DE整合图。方法:30例精子标本混匀后一次性进行蛋白提取,分别使用0.8、0.6、0.5、0.3 mg的蛋白上样量进行2-DE。运用MALD I-TOF法对确定2个恒定蛋白点的等位点(PI)和相对分子质量(Mr)作为图像的内参照点,对不同上样量的2-DE图进行比较分析,最后合成1张富集低丰度蛋白的整合图。结果:在0.5 mg图中分离出(1 080±23)个蛋白点,并合成具889个匹配点的整合图A。在上样量为0.8、0.6及0.3 mg的2-DE图中,分别新检测到381、50、32个在0.5 mg图中未检测到的蛋白点。最后合成具1 352个蛋白点的整合图B。结论:通过改变上样量的方法促进低丰度蛋白分离并合成具1 352个蛋白点富集低丰度蛋白的2-DE整合图。

Establishment of the 2-D Synthetic Map of Total Protein of Normal Human Spermatozoa Enriched with Low Abundance Protein

OBJECTIVE: To separate the low abundance protein and establish the 2-DE synthetic map of total protein of human normal spermatozoa by using the 2-DE technology. METHODS: All the needed human spermatozoa were collected and mixed, and proteins were extracted at one time with the method of urea/thiourea and ultra-sound. 0.8 mg, 0.6 mg, 0.5 mg, 0.3 mg sperm protein extracts were separated with 2-DE. Analyzed with MALDI-TOF-MS, PI and MW of 2 spots were obtained. Then set the 2 spots as the referent spots, different maps were compared and analyzed. At last, a synthetic map enriched with low abundance protein was obtained. RESULTS: 1,080 +/- 23 protein spots have been separated on the 2-DE map with standard 0.5 mg loading amount and a synthetic map A was constructed which consist of 889 matched protein spots on the all maps with 0.5 mg loading amount. 381, 50 and 32 new spots were detected individually on the maps with 0.8 mg, 0.6 mg and 0.3 mg protein loading amount. A synthetic map with 1,352 protein spots was obtained. CONCLUSION: Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount.