Analysis of seminal plasma proteomics for non-obstructive azoospermia by liquid chromatography tandem mass spectrometry
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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. -
图 1 差异表达蛋白火山图
A：原发性性腺功能减退组和性激素水平正常组差异蛋白火山图；B：继发性性腺功能减退组和性激素水平正常组差异蛋白火山图；C：原发性性腺功能减退组和继发性性腺功能减退组差异蛋白火山图
Figure 1. Volcano of DEPs
A: primary hypogonadism group vs normal sex hormone group; B: secondary hypogonadism group vs normal sex hormone group; C: primary hypogonadism group vs secondary hypogonadism group
图 2 原发性性腺功能减退组和性激素水平正常组差异蛋白GO富集分析
Figure 2. GO function analysis for DEPs of the primary hypogonadism group compared with the normal sex hormone group
图 3 原发性性腺功能减退组和性激素水平正常组差异蛋白KEGG通路
Figure 3. KEGG pathway analysis for DEPs of primary hypogonadism group compared with normal sex hormone group
图 4 继发性性腺功能减退组和性激素水平正常组差异蛋白GO富集
Figure 4. GO function analysis for DEPs of the secondary hypogonadism group compared with the normal sex hormone group
图 5 继发性性腺功能减退组和性激素水平正常组差异蛋白KEGG通路
Figure 5. KEGG pathway analysis for DEPs of the secondary hypogonadism group compared with the normal sex hormone group
图 6 原发性性腺功能减退组和继发性性腺功能减退组差异表达蛋白GO富集
Figure 6. GO function analysis for DEPs of the primary hypogonadism group compared with the secondary hypogonadism group
图 7 原发性性腺功能减退组和继发性性腺功能减退组差异表达蛋白KEGG通路
Figure 7. KEGG pathway analysis for DEPs of the primary hypogonad-ism group compared with the secondary hypogonadism group
图 8 差异表达蛋白Tissue Expression富集
B-C：继发性性腺功能减退组和性激素水平正常组；A-B：原发性性腺功能减退组和继发性性腺功能减退组。纵坐标为组间差异表达蛋白富集到该组织中的蛋白质数占总差异表达蛋白数的比例
Figure 8. Tissue expression analysis for DEPs
B-C: secondary hypogonadism group vs normal sex hormone group; A-B: primary hypogonadism group vs secondary hypogonadism group. The ordinate is the ration of the number of DEPs enriched into the tissue to the total number of DEPs
图 9 差异表达蛋白间相互作用(PPI)
橙色背景为上调蛋白(原发性性腺功能减退组＞继发性性腺功能减退组)；蓝色背景为下调蛋白(原发性性腺功能减退＜继发性性腺功能减退组)
Figure 9. Protein-protein interaction network of DEPs
Orange background: up-regulated proteins (primary hypogonadism group ＞ secondary hypogonadism group); Blue background: down-regulated proteins (primary hypogonadism group ＜ secondary hypogonadism group)
表 1 三组非梗阻性无精子症血清性激素水平
Table 1. Sera sex hormone levels in three groups of NOA
指标 PH组(n=12) SH组(n=6) NH组(n=3) 正常参考值 卵泡刺激素/(IU·L-1) 29.87 ± 7.24 0.76 ± 0.80 8.77 ± 3.94 1.4 ~ 18.1 黄体生成/(mIU·mL-1) 12.32 ± 8.93 0.34 ± 0.47 4.34 ± 1.27 1.5 ~ 9.3 睾酮/(nmol·L-1) 6.89 ± 4.22 2.46 ± 0.58 15.09 ± 5.37 8.4 ~ 28.7 性激素水平检测所用仪器为西门子全自动化学发光免疫分析仪，方法为化学发光法。 
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表 2 原发性性腺功能减退组和性激素水平正常组主要差异蛋白
Table 2. Details of the main DEPs in the primary hypogonadism group compared with normal sex hormone group
差异类型 注册号 蛋白质名称 基因符号 差异
倍数上调 P00441 Superoxide dismutase SOD1 19.86 P19652 Alpha-1-acid glycoprotein 2 ORM2 10.66 P20337 Ras-related protein Rab-3B RAB3B 8.89 Q99985 Semaphorin-3C SEMA3C 6.02 P13861 cAMP-dependent protein kinase type Ⅱ-alpha regulatory subunit PRKAR2A 5.81 H0YMD1 Low-density lipoprotein receptor LDLR 5.73 G8JLH6 Tetraspanin CD9 5.19 P09543 2',3'-cyclic-nucleotide 3'-phosphodiesterase CNP 4.86 Q687X5 Metalloreductase STEAP4 STEAP4 4.76 P08670 Vimentin VIM 4.67 P40926 Malate dehydrogenase, mitochondrial MDH2 4.66 O14786 Neuropilin-1 NRP1 4.14 Q9UIK5 Tomoregulin-2 TMEFF2 3.80 P34059 N-acetylgalactosamine-6-sulfatase GALNS 3.72 Q92930 Ras-related protein Rab-8B RAB8B 3.44 下调 P13645 Keratin, type Ⅰ cytoskeletal 10 KRT10 0.07 P55072 Transitional endoplasmic reticulum ATPase VCP 0.18 P13647 Keratin, type Ⅱ cytoskeletal 5 KRT5 0.23 P41218 Myeloid cell nuclear differentiation antigen MNDA 0.26 O95445 Apolipoprotein M APOM 0.31 Q9P2E9 Ribosome-binding protein 1 RRBP1 0.37 A0A087WXM9 Meiosis-specific kinetochore protein MEIKIN 0.42 O60547 GDP-mannose 4,6 dehydratase GMDS 0.49
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表 3 继发性性腺功能减退组和性激素水平正常组主要差异蛋白
Table 3. Details of the main DEPs in the secondary hypogonadism group compared with normal sex hormone group
差异类型 注册号 蛋白质名称 基因符号 差异
倍数上调 P22692 Insulin-like growth factor-binding protein 4 IGFBP4 29.61 P15907 Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 20.35 Q14894 Ketimine reductase mu-crystallin CRYM 9.48 Q92673 Sortilin-related receptor SORL1 7.98 P40926 Malate dehydrogenase, mitochondrial MDH2 7.31 Q92930 Ras-related protein Rab-8B RAB8B 6.98 P62873 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 GNB1 6.11 Q14520 Hyaluronan-binding protein 2 HABP2 5.57 Q9UNF0 Protein kinase C and casein kinase substrate in neurons protein 2 PACSIN2 4.32 Q96C19 EF-hand domain-containing protein D2 EFHD2 4.14 A0A0G2JRQ6 Ig-like domain-containing protein NA 3.20 Q9NSB4 Keratin, type Ⅱ cuticular Hb2 KRT82 2.92 P53041 Serine/threonine-protein phosphatase 5 PPP5C 2.59 P26885 Peptidyl-prolyl cis-trans isomerase FKBP2 FKBP2 2.16 下调 Q9Y6R7 IgGFc-binding protein FCGBP 0.02 O00468 Agrin AGRN 0.02 P08603 Complement factor H CFH 0.03 P06727 Apolipoprotein A-Ⅳ APOA4 0.04 P60174 Triosephosphate isomerase TPI1 0.04 B7ZKJ8 ITIH4 protein ITIH4 0.04 C9JV77 Alpha-2-HS-glycoprotein AHSG 0.05 P22314 Ubiquitin-like modifier-activating enzyme 1 UBA1 0.05 Q02818 Nucleobindin-1 NUCB1 0.05 P11021 Endoplasmic reticulum chaperone BiP HSPA5 0.05 P13489 Ribonuclease inhibitor RNH1 0.06 P14625 Endoplasmin HSP90B1 0.07 P02748 Complement component C9 C9 0.08 P62937 Peptidyl-prolyl cis-trans isomerase A PPIA 0.11 P16070 CD44 antigen CD44 0.20
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表 4 原发性性性腺功能减退组和继发性性腺功能减退组主要差异蛋白
Table 4. Details of the main DEPs in the primary hypogonadism group compared with secondary hypogonadism group
差异类型 注册号 蛋白质名称 基因符号 差异倍数 上调 Q9BWS9 Chitinase domain-containing protein 1 CHID1 104.40 P41182 B-cell lymphoma 6 protein BCL6 54.46 Q06210 Glutamine--fructose-6-phosphate aminotransferase GFPT1 34.79 Q8NCW5 NAD(P)H-hydrate epimerase NAXE 34.69 P08236 Beta-glucuronidase GUSB 34.47 Q06828 Fibromodulin FMOD 33.07 P16930 Fumarylacetoacetase FAH 25.31 P09488 Glutathione S-transferase Mu 1 GSTM1 21.57 P52209 6-phosphogluconate dehydrogenase PGD 21.13 P02748 Complement component C9 C9 12.00 P16070 CD44 antigen CD44 9.22 C9JV77 Alpha-2-HS-glycoprotein AHSG 5.68 B7ZKJ8 ITIH4 protein ITIH4 5.47 P14625 Endoplasmin HSP90B1 4.13 P62937 Peptidyl-prolyl cis-trans isomerase A PPIA 3.76 下调 Q92995 Ubiquitin carboxyl-terminal hydrolase 13 USP13 0.02 P02746 Complement C1q subcomponent subunit B C1QB 0.09 I3L486 Pancreatic secretory granule membrane major glycoprotein GP2 GP2 0.12 A0A087WTY6 Neuroblastoma suppressor of tumorigenicity 1 NBL1 0.12 P36507 Dual specificity mitogen-activated protein kinase kinase 2 MAP2K 0.12 Q6P4E1 Protein GOLM2 GOLM2 0.13 P40926 Malate dehydrogenase, mitochondrial MDH2 0.14 Q9UGM5 Fetuin-B FETUB 0.15 P31946 14-3-3 protein beta/alpha (Protein 1054) (Protein kinase C inhibitor protein 1) YWHAB 0.16 Q9NSB4 Keratin, type Ⅱ cuticular Hb2 KRT82 0.16 P26038 Moesin (Membrane-organizing extension spike protein) MSN 0.18 O75131 Copine-3 CPNE3 0.19 P01699 Immunoglobulin lambda variable 1-44 IGLV1-44 0.19 P04114 Apolipoprotein B-100 APOB 0.19 Q14520 Hyaluronan-binding protein 2 HABP2 0.19
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