Citation: Jingtian Lu, Ting Wang, Huimin Bao, Haojie Lu. Fractionation-free negative enriching for in-depth C-terminome analysis[J]. Chinese Chemical Letters, ;2022, 33(3): 1343-1345. doi: 10.1016/j.cclet.2021.08.022 shu

Fractionation-free negative enriching for in-depth C-terminome analysis

Jingtian Lu ^a ,
Ting Wang ^a ,
Huimin Bao ^a ,
Haojie Lu ^{a, b, *,}

a.
Department of Chemistry and Shanghai Cancer Center, Fudan University, Shanghai 200438, China
b.
Institutes of Biomedical Sciences and NHC Key Laboratory of Glycoconjugates Research, Fudan University, Shanghai 200032, China

luhaojie@fudan.edu.cn

Received Date: 23 April 2021
Revised Date: 4 August 2021
Accepted Date: 5 August 2021
Available Online: 11 August 2021

Figures(4)

Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, site-selective dimethylation and aldehyde resin coupling (MADMAR) for in-depth C-terminome analysis. The methylamidation blocked the free carboxyl group on proteins first, followed by LysC digestion of methylamidated proteins. Then, the site-selective dimethylation blocked the N-terminal amino group of the digested peptides without affecting the amino groups of lysine. Finally, the aldehyde resin was used to capture non-C-terminal peptides containing amino groups from lysine, while leaving the C-terminal peptides without free amino group in the supernatant for its analysis. We identified 1359 database-annotated protein C-termini from 50 µg HeLa proteins, which was 74% more than our previous method based on aldehyde resin. Moreover, 279 protein neo-C-termini were identified.
- C-terminome,
- In-depth analysis,
- Negative enrichment,
- Neo-C-termini,
- Methylamidation,
- HeLa
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