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

doi:10.13523/j.cb.2212033

China Biotechnology

2023, Vol. 43

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

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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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

Received: 23 December 2022 Published: 03 August 2023

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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.

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

Table 1 High performance liquid chromatography separation gradients

Fig.1 Experimental work flow chart and data quality control (a) After cold exposure, the cells of the two groups were collected and lysed. The protein in the cell lysate was digested with trypsin. Glycosylated peptides were digested with PNGas F. Then the methylated peptide was enriched with HILIC tip, and the protein methylation was ide.pngied on a large scale by LC-MS/MS (b) Pearson correlation analysis of methylated peptides

Fig.2 Data analysis of overall methylation sites (a) Ratio of methylation modification mode of lysine and arginine (b) Numbers of different methylation forms (c) Number of known and unknown methylated proteins ide.pngied (d) Blue: a known methylation site, gray: an unknown methylation site. The green bar indicates an unknown arginine methylation site, and the light brown bar indicates an unknown lysine methylation site (e) Sequence logo of ide.pngied and lysine (K) methylated sites within -6 to +6 amino acids, generated with WebLogo (f) Sequence logo of ide.pngied arginine (R) methylated sites within -6 to +6 amino acids

Fig.3 Bioinformatics analysis of methylated proteins (a) GO enriches methylated proteins. The filter condition for each annotation is P<0.05 (b) KEGG enrichment of ide.pngied methylated proteins. The filter condition for each pathway is P<0.01 (c) Mapping the methylated proteins onto the protein interaction network using the STRING database. Combine scores > 0.4

Fig.4 Differential analysis of methylated peptides and sites (a) Volcano plot illustrating the difference in abundance levels of methylation peptides. Log2(FoldChange) > 1 and P < 0.05 are the standard values of up-regulation conditions. Log2(FoldChange) < -1 and P<0.05 are the standard values of down-regulation conditions (b) Volcano plot illustrating the difference in abundance levels of methylation sites. The filter conditions are the same as in (a)


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