小鼠垂体瘤细胞冷暴露后蛋白质甲基化修饰分析<sup>*</sup>

doi:10.13523/j.cb.2212033

中国生物工程杂志

2023, Vol. 43

Issue (7): 12-22 DOI: 10.13523/j.cb.2212033

研究报告

小鼠垂体瘤细胞冷暴露后蛋白质甲基化修饰分析^*

徐颖^1,²,王雪²,王倩倩²,朱云平^1,^2,^**(

),贾辰熙^2,^**(

)

1 安徽医科大学基础医学院合肥 230032
2 蛋白质组学国家重点实验室国家蛋白质科学中心(北京) 北京蛋白质组研究中心北京生命组学研究所北京 102206

Analysis of Protein Methylation Modification of Mouse Pituitary Tumor Cells after Exposure to Cold

Ying XU^1,²,Xue WANG²,Qian-qian WANG²,Yun-ping ZHU^1,^2,^**(

),Chen-xi JIA^2,^**(

)

1 School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
2 State Key Laboratory of Proteomics, National Center for Protein Sciences (The PHOENIX Center, Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China

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摘要：

目的:研究小鼠垂体瘤细胞(mouse pituitary tumor cell,AtT20)冷暴露后蛋白质甲基化的变化,以及高效富集和大规模鉴定AtT20细胞的甲基化肽。方法:使用胰蛋白酶和肽N-糖苷酶F(peptide N-glycosidase F,PNGase F)消化蛋白质,利用亲水相互作用色谱(hydrophilic interaction liquid chromatography,HILIC)尖端富集甲基化肽,最后利用质谱法对甲基化肽进行整体分析;通过PhosphoSitePlus®数据库比对新的甲基化位点和蛋白,利用DAVID数据库进行GO功能富集和KEGG通路富集分析,最后对两组细胞所得的甲基化肽和甲基化位点进行差异分析。结果:共鉴定到55个甲基化蛋白和83个甲基化位点,其中78.3%的甲基化位点是未被报道过的;鉴定到的甲基化蛋白主要分布在核染色体、核小体和细胞核中,参与了基因沉默的调控、RNA剪接和mRNA加工等生物过程;KEGG通路富集表明这些蛋白与中性粒细胞胞外陷阱形成、酒精中毒和系统性红斑狼疮等相关。甲基化肽和甲基化位点差异分析显示有7条显著变化的甲基化肽和2个显著变化的位点。结论:AtT20细胞短期冷暴露后蛋白质甲基化变化不显著,首次对AtT20细胞进行全面甲基化蛋白质鉴定,发现了多个未被报道过的甲基化蛋白质和甲基化位点,以期为AtT20细胞的研究提供理论参考。

关键词： 小鼠垂体瘤细胞; 蛋白质甲基化; 冷暴露; 质谱

Abstract:

Objective: To study the changes of protein methylation in mouse pituitary tumor cell (AtT20) after exposure to cold, and to efficiently enrich and ide.pngy the methylated peptide of AtT20 cells. Methods: Trypsin and Peptide-N-Glycosidase F (PNGase F) were used to digest protein at the same time. Methylated peptides were enriched with the tip of hydrophilic interaction liquid chromatography (HILIC). Finally, the methylated peptides were analyzed by mass spectrometry. The PhosphoSitePlus® database was used to find new methylation sites and proteins, and the DAVID database was used to perform GO function enrichment and KEGG pathway enrichment analysis. Finally, the difference analysis was performed on the methylation peptides and methylation sites obtained from the two groups of cells. Results: 55 methylation proteins and 83 methylation sites were ide.pngied, of which 78.3% were unreported. The ide.pngied methylation proteins are mainly distributed in nuclear chromosomes, nucleosomes and nuclei, and they participate in the regulation of gene silencing, RNA splicing, mRNA processing and other biological processes. The enrichment of KEGG pathway indicates that these proteins are related to neutrophil exocytosis, alcoholism and systemic lupus erythematosus. The difference analysis of methylation peptides and methylation sites showed that there were 7 significantly changed methylation peptides and 2 significantly changed sites. Conclusion: The changes of protein methylation in AtT20 cells after short-term exposure to cold are not significant. For the first time, comprehensive ide.pngication of methylated proteins in AtT20 cells was carried out, and several unreported methylated proteins and methylation sites were found,which may provide new insights into the study of AtT20 cells.

Key words: Mouse pituitary tumor cell Protein methylation Cold exposure Mass spectrum

收稿日期: 2022-12-23 出版日期: 2023-08-03

ZTFLH:

Q816

基金资助: 国家重点研发计划(2021YFA1302601)

通讯作者: **电子信箱:zhuyunping@ncpsb.org.cn;cjia@ncpsb.org.cn

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引用本文:

徐颖, 王雪, 王倩倩, 朱云平, 贾辰熙. 小鼠垂体瘤细胞冷暴露后蛋白质甲基化修饰分析^*[J]. 中国生物工程杂志, 2023, 43(7): 12-22.

Ying XU, Xue WANG, Qian-qian WANG, Yun-ping ZHU, Chen-xi JIA. Analysis of Protein Methylation Modification of Mouse Pituitary Tumor Cells after Exposure to Cold. China Biotechnology, 2023, 43(7): 12-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2212033 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I7/12

表1 高效液相色谱分离梯度

图1 实验工作流程和数据质控

图2 总体甲基化位点数据分析

图3 甲基化蛋白质的生物信息学分析

图4 甲基化肽段和位点的差异分析

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