The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation
文献类型：期刊论文
| 作者 | Gao, Yuan3,4; Wang, Min3,4; Wang, Lulu3,4; Jia, Xinglong3; Hu, Chunqiu2,3; Liu, Ping3; Liu, Bin2; Tan, Minjia2,3,4 ; Zhai, Linhui1
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| 刊名 | JOURNAL OF MASS SPECTROMETRY
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| 出版日期 | 2025-06-01 |
| 卷号 | 60期号:6页码:12 |
| 关键词 | alkylation reaction quenching mass spectrometry pretreatment method proteomics |
| ISSN号 | 1076-5174 |
| DOI | 10.1002/jms.5141 |
| 英文摘要 | The reduction and alkylation steps are crucial in shotgun proteomics sample preparation to ensure efficient protein digestion and prevent the reformation of artefactual disulfide bonds following proteolysis. Excessive alkylation reagents can lead to overalkylation side reactions, compromising the quality of proteomics sample detection. Previous research has predominantly focused on comparing the effects of various types or concentrations of reducing agents or alkylating reagents for proteomic sample preparation. However, there is a lack of studies systematically comparing the utilization of quenching agents for alkylation reactions and investigating their specific impact on tryptic digestion activity in proteomics sample preparation under conditions of excessive alkylation reagents. In this study, we comprehensively compared the impacts of three different alkylation quenching methods (including cysteine quenching, dithiothreitol [DTT] quenching, and no quenching) on proteomic sample preparation. The upstream sample processing included reduction with DTT or tris(2-carboxyethyl)phosphine (TCEP), followed by alkylation with iodoacetamide (IAA) or chloroacetamide (CAA). Our study demonstrates that the choice of quenching method significantly affects the number of identified proteins and peptides, missed cleavage rates at lysine or arginine residues during trypsin digestion, and the occurrence of overalkylation side reactions. Importantly, our findings indicate that cysteine quenching effectively preserves trypsin activity, ensuring high-quality protein sample preparation. This study provides a systematic analysis of various alkylation quenching methods in proteomic sample preparation and offers optimized experimental protocols and valuable data references for proteomics studies. |
| WOS关键词 | MASS ; UBIQUITINATION ; REAGENT |
| 资助项目 | National Natural Science Foundation of China ; Program of Shanghai Academic Research Leader[22XD1420900] ; State Key Laboratory of Drug Research[SIMM2105KF-13] ; [32171434] ; [22225702] |
| WOS研究方向 | Biochemistry & Molecular Biology ; Chemistry ; Spectroscopy |
| 语种 | 英语 |
| WOS记录号 | WOS:001485659600001 |
| 出版者 | WILEY |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/317869]  |
| 专题 | 新药研究国家重点实验室 |
| 通讯作者 | Zhai, Linhui |
| 作者单位 | 1.Tongji Univ, Shanghai Peoples Hosp 4, Translat Res Inst Brain & Brain Like Intelligence, Sch Med, Shanghai, Peoples R China 2.Jiangsu Ocean Univ, Coll Pharm, Lianyungang, Jiangsu, Peoples R China 3.Chinese Acad Sci, State Key Lab Drug Res, Shanghai Inst Mat Med, Shanghai, Peoples R China 4.Nanjing Univ Chinese Med, Sch Chinese Mat Med, Sch Pharm, Nanjing, Jiangsu, Peoples R China |
| 推荐引用方式 GB/T 7714 | Gao, Yuan,Wang, Min,Wang, Lulu,et al. The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation[J]. JOURNAL OF MASS SPECTROMETRY,2025,60(6):12. |
| APA | Gao, Yuan.,Wang, Min.,Wang, Lulu.,Jia, Xinglong.,Hu, Chunqiu.,...&Zhai, Linhui.(2025).The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation.JOURNAL OF MASS SPECTROMETRY,60(6),12. |
| MLA | Gao, Yuan,et al."The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation".JOURNAL OF MASS SPECTROMETRY 60.6(2025):12. |
入库方式： OAI收割
来源：上海药物研究所
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