基于TMT技术的牦牛妊娠期血清蛋白质组学分析

doi:10.11843/j.issn.0366-6964.2024.01.019

摘要/Abstract

摘要： 为了探究妊娠期内牦牛血清的蛋白质组特征，筛选并分析差异表达蛋白质（DEPs）在牦牛妊娠维持中的作用。本研究选择9头4岁半至6岁龄健康状况良好且当年未产犊的自然发情母牦牛进行人工授精（AI）。在AI后第30、60及90天采集其血清样品，并对授配母牦牛进行妊娠诊断。依据诊断结果将采集的样品进行回顾性分组，即妊娠第1、2、3月组及未妊娠组（未妊娠组样品来自AI后90天经妊娠诊断确诊为未孕的牦牛个体），每组样品3个生物学重复。利用串联质谱标签（TMT）技术对牦牛血清样品的蛋白表达情况进行定量分析，筛选差异表达蛋白，并对其进行GO功能富集和KEGG通路等生物信息学分析，进一步筛选与牦牛妊娠维持相关的潜在蛋白。结果显示，在4组牦牛血清蛋白样品中共获得497个蛋白，以差异倍数≥1.2或≤0.83，且P<0.05为条件筛选，共筛选到68个差异蛋白。GO功能富集和KEGG通路分析显示，这些DEPs在代谢、免疫系统、粘附、生物调节等生物学过程以及补体与凝血级联、金黄色葡萄球菌感染等与炎症和免疫途径相关的通路有明显富集。值得注意的是，妊娠期各组间差异蛋白富集通路中均包含有PI3K-Akt、Rap1、MAPK等与生殖相关的信号通路。DEPs中IGFBP2、FGG、B2M、PDIA4、AZGP1的表达具有随妊娠时间的延长而连续上调的趋势，且这些蛋白在妊娠第3月表达量显著高于未妊娠状态。研究结果表明，牦牛妊娠早期存在明显的血清蛋白组的变化。这些DEPs分别在促进细胞粘附与增殖、母胎血管生成、维持母体代谢与免疫平衡等过程中起到重要作用。尤其是IGFBP2、FGG、B2M、PDIA4、AZGP1可能通过参与调节胎盘生长发育、免疫应答和脂质代谢等过程，在牦牛妊娠维持中发挥着重要作用。研究结果为进一步探明牦牛维持妊娠过程的调控机制提供了基础资料。

关键词: TMT, 蛋白质组学, 牦牛, 血清, 差异表达蛋白, 妊娠维持

Abstract: The purpose of this study was to explore the proteomic characteristics of yak serum during pregnancy, and to screen and analyze the role of differentially expressed proteins(DEPs) in the maintenance of yak pregnancy. In this study, nine natural estrus female yaks aged from 4.5 to 6 years old with good health and no calving in the current year were selected for artificial insemination(AI). Serum samples were collected on the 30, 60 and 90 days after AI, and pregnancy diagnosis was performed on the mated female yaks. According to the diagnostic results, the collected samples were retrospectively grouped into the first, second, third month of pregnancy group and non-pregnant group(non-pregnant group samples were from non-pregnant yak individuals diagnosed by pregnancy diagnosis 90 days after AI), with 3 biological replicates in each group. The protein expression of yak serum samples was quantitatively analyzed by tandem mass spectrometry(TMT) technology, and the differentially expressed proteins were screened. Bioinformatics analysis such as GO function enrichment and KEGG pathway were carried out to further screen potential proteins related to yak pregnancy maintenance. The results showed that a total of 497 proteins were obtained from the four groups of yak serum protein samples, and a total of 68 differential proteins were obtained by screening with a fold change ≥1.2 or ≤0.83, and P<0.05. GO functional enrichment and KEGG pathway analysis showed that these DEPs were significantly enriched in biological processes such as metabolism, immune system, adhesion, and biological regulation, as well as pathways related to inflammation and immune pathways such as complement and coagulation cascades and Staphylococcus aureus infection. It is worth noting that the differential protein enrichment pathways between the groups during pregnancy include PI3K-Akt, Rap1, MAPK and other reproductive-related signaling pathways. The expression of IGFBP2, FGG, B2M, PDIA4 and AZGP1 in DEPs had a tendency to be up-regulated with the prolongation of pregnancy time, and the expression of these proteins in the third month of pregnancy was significantly higher than that in the non-pregnant state. The results showed that there were obvious changes in serum proteome in early pregnancy of yak. These DEPs play an important role in promoting cell adhesion and proliferation, maternal-fetal angiogenesis, and maintaining maternal metabolism and immune balance. In particular, IGFBP2, FGG, B2M, PDIA4 and AZGP1 may play an important role in the maintenance of yak pregnancy by participating in the regulation of placental growth and development, immune response and lipid metabolism. The results provide basic data for further exploring the regulatory mechanism of yak in maintaining pregnancy.

Key words: TMT, proteomics, yak, serum, differentially expressed proteins, pregnancy maintenance

中图分类号:

S823.85

姚颖, 周应聪, 杜培岩, 李一娟, 钱文洁, 李柳杨, 余志鹏, 崔燕, 余四九, 樊江峰. 基于TMT技术的牦牛妊娠期血清蛋白质组学分析[J]. 畜牧兽医学报, 2024, 55(1): 192-206.

YAO Ying, ZHOU Yingcong, DU Peiyan, LI Yijuan, QIAN Wenjie, LI Liuyang, YU Zhipeng, CUI Yan, YU Sijiu, FAN Jiangfeng. Proteomic Analysis of Yak Serum During Pregnancy Based on TMT Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 192-206.

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