赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

引用本文: 李倩, 冯钰, 谭敏佳, 翟琳辉. 赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估[J]. 分析化学, 2017, 45(3): 316-321. doi: 10.11895/j.issn.0253-3820.160801 shu

Citation: LI Qian, FENG YU, TAN Min-Jia, ZHAI Lin-Hui. Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation[J]. Chinese Journal of Analytical Chemistry, 2017, 45(3): 316-321. doi: 10.11895/j.issn.0253-3820.160801 shu

赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

1.
中国科学院上海药物研究所新药研究国家重点实验室, 上海 201203;
2.
中国科学院大学, 北京 100049;
3.
北京华利世科技有限公司, 北京 101312
基金项目:
本文系上海市青年科技英才扬帆计划(No.16YF1414000)、国家自然科学基金(No.31370814)和上海市科学技术委员会(No.14DZ2261100)资助

摘要: 采用胰蛋白酶(Trypsin)单独酶切与不同酶量的赖氨酸C端内切酶(Lys-C/trypsin)顺序酶切两种方法，对293T细胞全蛋白样本进行酶解消化，系统评估Lys-C/trypsin顺序酶切与Trypsin单一酶切在蛋白质组学样本制备中的差别。实验结果表明，Lys-C/trypsin顺序酶切不仅能显著提高肽段和蛋白质的鉴定数目，同时降低遗漏K酶切位点的数目及比例，而且得到的肽段长度有利于质谱鉴定，蛋白质覆盖率明显提升。通过对酶的用量进行优化对比，最终确定了Lys-C/trypsin顺序酶切时酶的合理用量。本研究结果对提高蛋白质组学样本的制备质量以及蛋白质的序列鉴定覆盖度具有指导意义。

关键词:

English

Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation

1.
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;
2.
University of Chinese Academy of Sciences, Beijing 100049, China;
3.
Beijing Hualishi Technology Co., Ltd., Beijing 101312, China
Received Date: 02 November 2016
Revised Date: 23 December 2016
Fund Project:
This work was supported by Youth Science and Technology Talents in Shanghai Sailplan (No. 16YF141400) the National Natural Science Foundation of China (No.31370814) and Shanghai Science and Technology Committee (No.14DZ2261100)

Abstract: Endoproteinase Lys-C/trypsin sequential digestion and trypsin digestion were used in 293T cell proteomics sample preparation and the results of Lys-C/trypsin sequential digestion and trypsin digestion in proteomics sample preparation was systematically evaluated. It was found that the number of identified peptides and proteins increased significantly, and missed cleavage sites, especially K sites decreased dramatically through Lys-C/trypsin sequential digestion. And the average sequence coverage of identified proteins in Lys-C/trypsin sequential digestion sample was higher than that in trypsin digestion sample. Besides, different amount of enzymes was tested to select the optimal usage of enzymes in Lys-C/trypsin sequential digestion. This study provided the references for proteomics sample preparation.

Key words:

Trypsin
/ Lys-C
/ Sequential digestion
/ Proteomics
/ Liquid chromatography-tandem mass spectrometry
/ Missed cleavage

1. [1]
  Malmström E, Kilsgård O, Hauri S, Smeds E, Herwald H, Malmström L, Malmström J. Nat. Commun., 2016,7:10261-10270
2. [2]
  Link A J, Eng J, Schieltz D M, Carmack E, Mize G J, Morris D R, Garvik B M, Yates J R. Nat. Biotechnol., 1999,17(7):676-682
3. [3]
  de Godoy L M, Olsen J V, Cox J, Nielsen M L, Hubner N C, Mann M. Nature, 2008,455(7217):1251-1254
4. [4]
  Wilhelm M, Schlegl J, Hahne H, Gholami A M, Lieberenz M, Butzmann L, Gerstmair A, Faerber F, Kuster B. Nature, 2014,509(7502):582-587
5. [5]
  Zhang B, Wang J, Wang X, Zhu J, Liu Q, Shi Z, Chambers M C, Davies S R, Coffey R J, Slebos R J, Liebler D C. Nature, 2014,513(7518):382-387
6. [6]
  Lawrence R T, Perez E M, Hernandez D, Miller C P, Haas K M, Irie H Y, Lee S I, Blau C A, Villen J. Cell. Rep., 2015,11(4):630-644
7. [7]
  Lawrence R T, Perez E M, Hernandez D, Miller C P, Haas K M, Irie H Y, Lee S I, Blau C A, Villen J. Nat. Commun., 2016,7:12645
8. [8]
  Tsiatsiani L, Heck A J. FEBS. J., 2015,282(14):2612-2626
9. [9]
  Saveliev S, Bratz M, Zubarev R. Nat. Methods, 2013,10(11):i-ii
10. [10]
  Huesgen P F, Lange P F, Rogers L D. Nat. Methods, 2015,12(1):55-58
11. [11]
  Link A J, Eng J, Schieltz D M, Carmack E, Morris D R, Garvik B M, Yates J R. Nat. Biotechnol., 1999,17(7):676-682
12. [12]
  McDonald W H, Ohi R, Miyamoto D T, Mitchison T J, Yates J R. Int. J. Mass. Spectrom., 2002, 219:245-251
13. [13]
  Wada Y, Kadoya M. J. Mass. Spectrom., 2003,38(1):117-118
14. [14]
  Wisniewski J R, Mann M. Anal. Chem., 2012,84(6):2631-2367
15. [15]
  Wisniewski J R, Zougman A, Nagaraj N, Mann M. Nat. Methods, 2009,6(5):359-362
16. [16]
  Scheltema R A, Hauschild J P, Lange O, Makarov A, Mann M. Mol. Cell. Proteomics, 2014,13(12):3698-3708
17. [17]
  Glatter T, Ludwig C, Ahrné E, Aebersold R, Heck A J, Schmidt A. J. Proteome Res., 2012,11(11):5145-5156
18. [18]
  Mertins P, Mani D R, Ruggles K V, Gillette M A, Paulovich A G, Fenyo D, Ellis M J, Carr S A. Nature, 2016,534(7605):55-62
19. [19]
  Cox J, Hein M Y, Luber C A, Paron I, Nagaraj N, Mann M. Mol. Cell. Proteomics, 2014,13(9):2513-2526
20. [20]
  Paulo J A, Gigy S P. Proteomics, 2015,15(23):474-486
21. [21]
  Hebert A S, Richards A L, Coon J J. Mol. Cell. Proteomics, 2014,13(1):339-347

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赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

English

Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation

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中国化学会秘书处

赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

English

Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

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CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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