伴刀豆球蛋白A诱导肝细胞损伤的磷酸化蛋白质组学研究

doi:10.3969/j.issn.1674-8115.2021.01.001

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (1): 1-7.doi: 10.3969/j.issn.1674-8115.2021.01.001

伴刀豆球蛋白A诱导肝细胞损伤的磷酸化蛋白质组学研究

杨卓一¹(), 陈会¹, 白思益¹, 帕梅拉·帕尔哈提null¹, 侯敬丽², 袁运生¹()

^1.上海交通大学药学院细胞工程及抗体药物教育部研究工程中心，上海 200240
^2.上海交通大学分析测试中心，上海 200240

出版日期:2021-01-28 发布日期:2021-02-22
通讯作者: 袁运生 E-mail:yangzhuoyi@sjtu.edu.cn;yunsheng@sjtu.edu.cn
作者简介:杨卓一（1993—），女，硕士生；电子信箱：yangzhuoyi@sjtu.edu.cn。
基金资助:
国家科技重大专项(2019ZX09201001);国家自然科学基金(31671388);上海交通大学医工交叉项目(YG2019QNA50)

Phosphoproteomic analysis of concanavalin A-induced hepatocytes injury

Zhuo-yi YANG¹(), Hui CHEN¹, Si-yi BAI¹, Pameila PERHATI¹, Jing-li HOU², Yun-sheng YUAN¹()

^1.Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education; Shanghai Jiao Tong University School of Pharmacy, Shanghai 200240, China
^2.Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China

Online:2021-01-28 Published:2021-02-22
Contact: Yun-sheng YUAN E-mail:yangzhuoyi@sjtu.edu.cn;yunsheng@sjtu.edu.cn
Supported by:
Funding Information] National Science and Technology Major Project(2019ZX09201001);National Natural Science Foundation of China(31671388);Shanghai Jiao Tong University Medical-Engineering Joint Project(YG2019QNA50)

摘要/Abstract

摘要：

目的·研究伴刀豆球蛋白A（concanavalin A，ConA）诱导肝细胞损伤过程中全细胞蛋白质的磷酸化修饰水平的变化规律。方法·将小鼠肝细胞株AML12与ConA（10 μg/mL）共培养，诱导肝细胞损伤模型，12 h后提取细胞的总蛋白，经消化酶解后富集肽段，并使用高效液相-质谱联用分析仪进行检测。采用非标定量法对磷酸化位点和肽段进行鉴定与相对定量分析。筛选出单位点磷酸化修饰水平差异达到2倍以上的蛋白进行生物学功能富集分析和蛋白互作调控网络分析。结果·在ConA诱导的AML12细胞损伤模型中，共鉴定到磷酸化修饰肽段11 200条，可定位定量的肽段2 685条。其中差异性调控的磷酸化修饰肽段82条，对应77个蛋白。这77个磷酸化蛋白主要与蛋白质结合、酶活动等相关，且主要通路与细胞凋亡以及RAS激酶通路相关。结论·AML12细胞在ConA诱导的肝细胞损伤过程中发生了磷酸化修饰变化，差异磷酸化修饰的蛋白可能与肝细胞凋亡相关。

关键词: 伴刀豆球蛋白A, AML12细胞株, 蛋白质组学, 磷酸化修饰

Abstract:

Objective ·To study the changes of phosphorylation level of whole-cell proteins during the process of concanavalin A (ConA)-induced hepatocyte injury.

Methods ·Mouse hepatocytes of AML12 cells and ConA (10 μg/mL) were co-cultured to induce a hepatocyte injury model. After 12 h, the total proteins were extracted and digested into peptides. The peptides were enriched by metal oxide affinity chromatography and detected by using high performance liquid chromatography-mass spectrometer. Identification and relative quantitative analysis of phosphorylation sites and peptides were performed using the Label free method. Proteins with a difference of more than two times of phosphorylation level of each site were screened. And the biological function enrichment analysis and protein interaction regulation network analysis were performed.

Results ·In the ConA-induced AML12 cell injury model, a total of 11 200 phosphorylated modified peptides and 2 685 localizable and quantifiable peptides were identified. Among them, there were 82 differentially regulated phosphorylated modified peptides, corresponding to 77 proteins. These phosphorylated proteins were mainly related to protein binding and enzyme activities. And the main pathways were related to apoptosis and RAS kinase pathways.

Conclusion ·AML12 cells undergo phosphorylation level change in ConA-induced liver cell injury and these differentially phosphorylated proteins may be related to liver cell apoptosis.

Key words: concanavalin A (ConA), AML12 cell line, proteomics, phosphorylation modification

中图分类号:

R915

杨卓一, 陈会, 白思益, 帕梅拉·帕尔哈提null, 侯敬丽, 袁运生. 伴刀豆球蛋白A诱导肝细胞损伤的磷酸化蛋白质组学研究[J]. 上海交通大学学报(医学版), 2021, 41(1): 1-7.

Zhuo-yi YANG, Hui CHEN, Si-yi BAI, Pameila PERHATI, Jing-li HOU, Yun-sheng YUAN. Phosphoproteomic analysis of concanavalin A-induced hepatocytes injury[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(1): 1-7.

图/表 5

图1 差异磷酸化蛋白质组学的鉴定及定量结果Note:A. Results of the distribution of phosphorylation sites in proteins. B. Phosphorylation modification ratio histogram. C. Heatmap of differentially phosphorylated peptides.

Fig 1 Results of identification and quantification of differentially phosphorylated proteomics

表1 前10个差异磷酸化修饰肽段序列和修饰位点信息

Tab 1 Top ten differentially phosphorylated peptide sequences and modification site information

No.	Uniport ID	Protein name	Sequence of peptide	Modification site	Position on protein
1	P99029	Prdx5	[K].RFSMVIDNGIVK.[A]	[S3]	[S178]
2	Q9JLN9	mTOR	[K].LHVSTINLQK.[A]	[S4]	[S1 261]
3	Q9EST3	eIF4E	[R].APSPPMSQVFR.[T]	[S3]	[S563]
4	Q9JJT9	Phax	[K].CHNTPPKPEPFPFGPSGQK.[T]	[T4]	[T80]
5	Q80XP8	Fam76b	[K].VSSLSPEQEQGLWK.[Q]	[S3]	[S191]
6	Q9D0F9	Pgm1	[K].AIGGIILTASHNPGGPNGDFGIK.[F]	[S10]	[S117]
7	Q52KI8	Srrm1	[R].HRPSSPATPPPK.[T]	[S5; T8]	[S401; T404]
8	Q05D44	eIF5B	[R].DGSEEDEDNSKR.[S]	[S3]	[S165]
9	Q9R078	Prkab1	[R].SQNNFVAILDLPEGEHQYK.[F]	[S1]	[S108]
10	Q99JG3	Anxa13	[K].ERSHHHGFDADR.[D]	[S3]	[S10]

图2 mTOR磷酸化修饰肽段二级质谱和2组间含量比较结果Note:A. Secondary mass spectrum of mTOR phosphorylation modified peptides. B. Comparison of total mTOR phosphorylated peptides. ①P=0.049 5, compared with the control group.

Fig 2 Secondary mass spectrum of mTOR phosphorylation modified peptides and comparison between the two groups

图3 差异磷酸化修饰蛋白GO分析结果Note:A. Differentially phosphorylated protein-related biological processes. B. Differentially phosphorylated protein-involved molecular interactions.

Fig 3 Results of GO analysis of differentially phosphorylated modified proteins

图4 差异磷酸化修饰蛋白质互作关系网络图

Fig 4 Interaction network of differentially phosphorylated modified proteins

[1]	黄原昕, 赖东梅. 蛋白质组学技术在妇科疾病研究中的应用[J]. 上海交通大学学报(医学版), 2021, 41(2): 233-240.
[2]	熊强强, 屠俊, 李郡如, 程金科, 左建宏, 陈亚兰. 蛋白质SUMO化修饰的蛋白质组学研究述评[J]. 上海交通大学学报(医学版), 2021, 41(1): 89-94.
[3]	刘锦燕，史册，王影，李文静，赵悦，朱巍巍，项明洁 . 蛋白质组学在白假丝酵母研究中的应用[J]. 上海交通大学学报（医学版）, 2016, 36(12): 1804-.
[4]	胡江，闻大翔，杭燕南. 自发性高血压大鼠丙泊酚麻醉后海马组织的蛋白质组学研究[J]. 上海交通大学学报（医学版）, 2014, 34(4): 491-.
[5]	李潺, 胡江, 闻大翔, 等. 丙泊酚对糖尿病大鼠海马组织蛋白质表达影响的蛋白组学研究[J]. , 2011, 31(6): 748-.
[6]	彭立威, 杨国锋, 纪建国, 等. 急性运动轴索型神经病患者血清对大鼠脊髓运动神经元凋亡相关蛋白表达的影响[J]. , 2011, 31(5): 580-.
[7]	张毅, 苗玲, 苏敏, 等. 神经细胞老化过程中蛋白质组学研究[J]. , 2010, 30(4): 432-.

伴刀豆球蛋白A诱导肝细胞损伤的磷酸化蛋白质组学研究

Phosphoproteomic analysis of concanavalin A-induced hepatocytes injury

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