Citation: YANG Chao, LIU Jian-Hui, ZHANG Wei-Jie, SHAN Yi-Chu, DAI Zhong-Peng, ZHANG Li-Hua, ZHANG Yu-Kui. Peptides De Novo Sequencing Strategy Based on Pseudo Isobaric Peptide Termini Labeling[J]. Chinese Journal of Analytical Chemistry, ;2021, 49(3): 366-376. doi: 10.19756/j.issn.0253-3820.201627 shu

Peptides De Novo Sequencing Strategy Based on Pseudo Isobaric Peptide Termini Labeling

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CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
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University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding author: SHAN Yi-Chu, shanyichu@dicp.ac.cn
Received Date: 26 October 2020
Revised Date: 29 December 2020

Fund Project: National key Research and Development Program of China (Nos. 2017YFF0205404, 2017YFA0505004) and the National Natural Science Foundation of China (No.21675153).

Peptide de novo sequencing is of great significance for the functional study of active peptides, discovery of disease-related protein mutants and the quality control of monoclonal antibodies. The peptide de novo sequencing method determines the types of amino acid residues based on the mass differences of ions in the mass spectrum, and then derives the amino acid sequence of the peptide. Due to the presence of noise and interfering ions in mass spectrum, the accuracy of sequencing by current sequencing algorithms is low. In this study, a de novo sequencing method based on pseudo-isobaric peptide termini labeling (PIPTL) was developed. The peptides were divided equally into two parts. For the first part, the N-termini of the peptide was dimethylated with formaldehyde, and the ¹⁶O of the C-terminal carboxyl group was replaced with ¹⁸O.For the other part, the N-termini was dimethylated with deuterated formaldehyde. The two labeled peptide samples were mixed in equal. Different labeled peptides of the same sequence differ in precursor mass by only 0.016 Da. These pseudo isobaric peptides could be fragmented at the same time in the mass spectrum to produce paired fragment ions. The developed sequencing algorithm simplified the spectra and effectively extracted and distinguished b/y ions and sequence peptides. This strategy had an accuracy of 95.51% for sequencing tryptic digested bovine serum albumin. The sequencing accuracy was significantly higher than the commercial sequencing software PEAKS. In addtion, the monoclonal antibody Herceptin tryptic peptides were sequenced, and the sequencing accuracy was 93.60%. This method could be applied to the field of characterization of monoclonal antibody sequences.
- Peptide sequencing,
- De novo sequencing,
- Pseudo isobaric labeling
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沈阳化工大学材料科学与工程学院沈阳 110142