Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of optimized sample preparation methods to achieve in-depth protein coverage for sperm cells. Meanwhile, it is not clear whether antibiotics can regulate proteins to affect sperm quality. Here, we systematically compared a total of six different protein extraction methods based the combination of three commonly used lysis buffers and physical lysis strategies. The urea buffer combined with ultrasonication (UA_ultrasonication) produced the highest protein extraction rate, leading to the deepest coverage of human sperm proteome (5685 protein groups) from healthy human sperm samples. Since the antibiotics, amoxicillin and clarithromycin, have been widely used against H. pylori infection, we conduct a longitudinal study of sperm proteome via data-independent acquisition tandem mass spectrometry (DIA-MS/MS) on an infected patient during on and off therapy with these two drugs. The semen examination and morphological analysis were performed combined with proteomics analysis. Our results indicated that antibiotics may cause an increase in the sperm concentration and the rate of malformed sperm and disrupt proteome expression in sperm. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications.

中文翻译：

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人类精子蛋白质组图谱：优化提取方法及应用

正在探索暴露于某些物理、生物或化学应激源的人类精子蛋白质组学。然而，缺乏优化的样品制备方法来实现精子细胞的深度蛋白质覆盖。同时，尚不清楚抗生素是否可以通过调节蛋白质来影响精子质量。在这里，我们基于三种常用的裂解缓冲液和物理裂解策略的组合，系统地比较了总共六种不同的蛋白质提取方法。尿素缓冲液结合超声波处理 (UA_ultrasonication) 产生了最高的蛋白质提取率，导致健康人类精子样本中人类精子蛋白质组（5685 个蛋白质组）的最深覆盖。由于抗生素阿莫西林和克拉霉素已被广泛用于治疗幽门螺杆菌感染，我们通过数据独立采集串联质谱法 (DIA-MS/MS) 对一名感染患者在使用这两种药物进行断断续续的治疗期间对精子蛋白质组进行了纵向研究。精液检查和形态学分析与蛋白质组学分析相结合。我们的结果表明，抗生素可能会导致精子浓度和畸形精子发生率增加，并破坏精子中蛋白质组的表达。这项工作提供了一种优化的提取方法来表征深入的人类精子蛋白质组并扩展其临床应用。我们的结果表明，抗生素可能会导致精子浓度和畸形精子发生率增加，并破坏精子中蛋白质组的表达。这项工作提供了一种优化的提取方法来表征深入的人类精子蛋白质组并扩展其临床应用。我们的结果表明，抗生素可能会导致精子浓度和畸形精子发生率增加，并破坏精子中蛋白质组的表达。这项工作提供了一种优化的提取方法来表征深入的人类精子蛋白质组并扩展其临床应用。