轩冉, 薛丹丹, 王春杨, 鲍金凤, 虞亚菲, 段晋燕. 非梗阻性无精子症患者精浆蛋白质组学研究[J]. 解放军医学院学报, 2023, 44(5): 514-524. DOI: 10.3969/j.issn.2095-5227.2023.05.013
引用本文: 轩冉, 薛丹丹, 王春杨, 鲍金凤, 虞亚菲, 段晋燕. 非梗阻性无精子症患者精浆蛋白质组学研究[J]. 解放军医学院学报, 2023, 44(5): 514-524. DOI: 10.3969/j.issn.2095-5227.2023.05.013
XUAN Ran, XUE Dandan, WANG Chunyang, BAO Jinfeng, YU Yafei, DUAN Jinyan. Analysis of seminal plasma proteomics for non-obstructive azoospermia by liquid chromatography tandem mass spectrometry[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(5): 514-524. DOI: 10.3969/j.issn.2095-5227.2023.05.013
Citation: XUAN Ran, XUE Dandan, WANG Chunyang, BAO Jinfeng, YU Yafei, DUAN Jinyan. Analysis of seminal plasma proteomics for non-obstructive azoospermia by liquid chromatography tandem mass spectrometry[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(5): 514-524. DOI: 10.3969/j.issn.2095-5227.2023.05.013

非梗阻性无精子症患者精浆蛋白质组学研究

Analysis of seminal plasma proteomics for non-obstructive azoospermia by liquid chromatography tandem mass spectrometry

  • 摘要:
      背景  非梗阻性无精子症(non-obstructive azoospermia,NOA)是男性不育患者中十分严重的一种情况,目前其病理生理学机制未完全明确。
      目的  应用液相色谱串联质谱(LC-MS/MS)定量蛋白组学技术分析非梗阻性无精子症NOA患者精浆蛋白组学,初步探索NOA相关的候选生物标志物。
      方法  纳入2020年10月 - 2022年1月解放军总医院第一医学中心门诊行精液常规分析的NOA患者21例,按性激素水平分为原发性性腺功能减退(primary hypogonadism,PH)组(12例)、继发性性腺功能减退(secondary hypogonadism,SH)组(6例)和性激素水平正常组(NH组,3例),使用Label-free质谱技术对患者精浆进行蛋白质鉴定和相对定量。采用Proteome Discoverer软件对结果进行搜库鉴定,并对差异蛋白进行生物信息学分析。
      结果  在检测到的1144种蛋白质中,与性激素水平正常组相比,原发性性腺功能减退组有32个上调差异蛋白和8个下调差异蛋白,继发性性腺功能减退组有14上调差异蛋白和99个下调差异蛋白;与继发性性腺功能减退组相比,原发组有146个上调差异蛋白,21个下调差异蛋白。差异表达蛋白功能主要包括酶活性、代谢、肽链内切酶调节活性、受体结合,主要富集到溶酶体、补体和凝血级联、糖降解、氨基酸代谢等通路。蛋白质-蛋白质相互作用网络显示,苹果酸脱氢酶2 (malate dehydrogenase 2,MDH2)、肽酰-脯氨酰顺反异构酶A (peptidyl-prolyl cis-trans isomerase A,PPIA)、α-胰蛋白酶间抑制剂重链H4(inter-alpha-trypsin inhibitor heavy chain H4,ITIH4)、热休克蛋白90B1 (heat shock protein 90 beta1,HSP90B1)、α-2-HS-糖蛋白(alpha-2-HS-glycoprotein,AHSG)、CD44抗原和补体C9可能在NOA患者精子生成调控网络中发挥重要作用,其中重点分析了MDH2和HSP90B1。与继发性性腺功能减退组相比,原发组MDH2下调,HSP90B1上调。
      结论  本研究结果表明,MDH2、PPIA、ITIH4、HSP90B1、AHSG、CD44和C9可能在NOA患者精子生成调控网络中发挥重要作用。MDH2低表达提示睾丸功能异常致NOA,HSP90B1低表达通过调节脂质代谢影响性激素水平致NOA。差异表达蛋白的功能富集分析提示氨基酸代谢和溶酶体消化功能异常可能会引起下丘脑-垂体-性腺轴正负反馈调节紊乱;代谢途径异常、糖降解紊乱、补体和凝血级联紊乱可能会导致睾丸功能障碍。

     

    Abstract:
      Background  Non-obstructive azoospermia (NOA) is the most serious condition in male infertility patients, and its pathophysiology remains unclear.
      Objective  To preliminary investigate the candidate biomarkers of NOA and analyze seminal plasma of NOA by liquid chromatography with tandem mass spectrometry (LC-MS/MS) quantitative proteomics.
      Methods  Totally 21 NOA patients who underwent semen routine analysis in the First Medical Center of Chinese PLA General Hospital from October 2020 to January 2022 were recruited, and they were divided into primary hypogonadism group (PH group, n=12), secondary hypogonadism group (SH group, n=6) and normal sex hormone group (NH group, n=3). Identification and relative quantification of proteins using label-free proteomics via LC-MS/MS was carried out on seminal plasma from NOA patients. MS data files were imported to Proteome Discoverer software to identify the proteins. Then bioinformatics analysis of differentially expressed proteins (DEPs) was performed.
      Results  A total of 1 144 proteins were detected. Compared with group NH, there were 32 up-regulated DEPs and 8 down-regulated DEPs in PH group, 14 up-regulated DEPs and 99 down-regulated DEPs in SH group. Compared with SH group, 146 up-regulated DEPs and 21 down-regulated DEPs were found in PH group. The functions of DEPs mainly included hydrolase activity, enzyme catalytic activity, metabolism, peptide endonuclease regulation activity and receptor binding GO terms, and the pathways of DEPs mainly enriched into lysosome, complement and coagulation cascade, glucose degradation and amino acid metabolism pathways. Protein-protein interaction network by STRING revealed that Malate dehydrogenase (MDH2), Peptidyl-prolyl cis-trans isomerase (PPIA), Inter-alpha-trypsin inhibitor heavy chain (ITIH4), Heat shock protein 90 kDa (HSP90B1), Alpha-2-HS-glycoprotein (AHSG), CD44 antigen (CD44) and Complement component (C9) might play important roles in the spermatogenesis regulation network in NOA, and we focused on two proteins, MDH2 and HSP90B1, which were associated with spermatogenesis. Compared with SH group, MDH2 was down-regulated and HSP90B1 was up-regulated in PH group.
      Conclusion  In conclude, MDH2, PPIA, ITIH4, HSP90B1, AHSG, CD44 and C9 may play important roles in the spermatogenesis regulation network in NOA. Moreover, low expression of MDH2 is caused by testicular dysfunction leading to NOA, while the low expression of HSP90B1 is caused by regulating lipid metabolism affecting sex hormone levels. The functional enrichment analysis of DEPs suggests that the abnormal amino acid metabolism and lysosome digestion may contribute to disorder of hypothalamic-pituitary-gonad axis negative and positive feedback regulation. Abnormal metabolism pathway, disrupted glucose degradation, and disrupted complement and coagulation cascade may lead to testicular dysfunction.

     

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