Effects of Different Sequential Enzymatic Cleavage of Trypsin and LysC on Proteomic Sample Preparation
10.13865/j.cnki.cjbmb.2024.09.1220
- VernacularTitle:胰蛋白酶与赖氨酸C端内切酶不同顺序酶切对蛋白质组样本制备的影响
- Author:
Rui-Dong LI
1
,
2
;
Min WANG
;
Lu-Lu WANG
;
Ming-Ya ZHANG
;
Yuan GAO
;
Min-Jia TAN
;
Fang GUO
;
Lin-Hui ZHAI
Author Information
1. 江苏海洋大学药学院,江苏省海洋药物化合物筛选重点实验室,江苏 连云港222005
2. 中国科学院上海药物研究所药物研究国家重点实验室,上海201203
- Keywords:
proteomics;
lysine C-terminal endonuclease (LysC);
trypsin;
in-solution digestion;
sequential digestion
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(11):1618-1626
- CountryChina
- Language:Chinese
-
Abstract:
In mass spectrometry-based proteomics experiments,achieving high-throughput and efficientproteolytic digestion is crucial to ensure optimal protein cleavage and enhance the depth of protein identi-fication (including the number of identified proteins and the coverage of protein amino acid sequences) .Trypsin is the most widely used protease in mass spectrometry-based proteomics due to its ability to spe-cifically cleave the carboxyl terminus of arginine and lysine.However,it was found that Trypsin has some missed enzymatic efficiency for the cleavage of lysine residues.Therefore,in actual proteomics sample preparation,a combination of Trypsin and LysC will be used to ensure adequate cleavage of lysine resi-dues.Our study revealed that the commonly employed LysC-Trypsin tandem cleavage method exerts an impact on the enzymatic cleavage of protein samples by Trypsin due to the subsequent cleavage of Trypsin by initially added LysC.Consequently,we adjusted the order of LysC and Trypsin tandem digestion,with Trypsin cleavage being performed first followed by the addition of LysC to target any missed lysine resi-dues.We comprehensively compared and analyzed three distinct sequential digestion methods,namely Trypsin-Trypsin (T-T),LysC-Trypsin (L-T),and Trypsin-LysC (T-L),in terms of their effects on pro-tein sample preparation quality.The results demonstrated that the Trypsin-LysC sequential digestion ap-proach not only minimizes missed protein lysine/arginine cleavage sites without increasing experimental costs,at the same time yielding peptides with a moderate amino acid sequence length.The use of Tryp-sin-LysC digestion enhances the adsorption and separation of peptide samples in RP-HPLC,as well as improves the depth of protein detection and amino acid sequence coverage during tandem mass spectrome-try analysis.This research work offers a novel technical solution and serves as a valuable reference for proteome sample preparation.
