The present study is a contribution to the “neXt50 challenge”, a coordinated effort across C-HPP teams to identify the 50 most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass spectrometry-based strategy consisting in the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with 2 or more peptides and 3 with 1 peptide after extensive SDS-PAGE fractionation of the two samples and with overall low intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community as an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.

中文翻译：

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通过靶向质谱法和基于抗体的方法验证人精子细胞中缺失的蛋白质

本研究为“ neXt50挑战”做出了贡献，“ neXt50挑战”是C-HPP团队之间的协同努力，旨在鉴定每个染色体上最容易处理的50种缺失蛋白（MPs）。我们报告了针对共有15个健康供体的Triton X-100可溶和不可溶精子级分中2号和14号染色体上38个理论上可检测的MP的目标搜索。使用了基于目标质谱的策略，该策略包括开发针对38个选定MP中92个蛋白型肽的LC-PRM分析（带有重标记的合成肽）。在两个样品进行广泛的SDS-PAGE分级分离后，从38个选定的MP中鉴定出12个具有2个或更多的肽，3个具有1个肽，并且总体强度低。可通过PASSEL（PASS01013）中的ProteomeXchange获得PRM数据。用人类蛋白图谱中的抗体对八种MP进行了进一步的验证，方法是对人体睾丸切片进行免疫组织化学，对精子涂片进行细胞化学检查。对人类精子的深入分析仍然可以验证MP，因此有助于C-HPP在全球范围内的努力。我们预计我们的结果将对生殖生物学界产生兴趣，因为对这些MP的深入分析可能会在人类特发性不育症的背景下确定潜在的新候选人。