基于末端准等重同位素标记的肽段从头测序方法  

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

作　　者：杨超 刘健慧 张玮杰 单亦初[1] 戴忠鹏[1] 张丽华[1] 张玉奎[1] YANG Chao;LIU Jian-Hui;ZHANG Wei-Jie;SHAN Yi-Chu;DAI Zhong-Peng;ZHANG Li-Hua;ZHANG Yu-Kui(CAS Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,大连116023 [2]中国科学院大学,北京100049

出　　处：《分析化学》2021年第3期366-376,共11页Chinese Journal of Analytical Chemistry

基　　金：国家重点研发计划项目(Nos.2017YFF0205404,2017YFA0505004);国家自然科学基金项目(No.21675153)资助。

摘　　要：蛋白质的从头测序在蛋白质生物功能的研究、疾病相关蛋白突变体的发现和抗体药物表征等领域具有关键作用,其主要方法是将蛋白质酶解成肽段,通过二级谱图中碎片离子的质量差异判断氨基酸残基的序列信息,进而实现肽段的从头测序。由于谱图中的碎片离子信号易受噪声和不能用于测序的离子的干扰,目前的从头测序算法准确度较低。本研究发展了一种基于肽段末端准等重同位素标记(Pseudo-isobaric peptide termini labeling,PIPTL)的从头测序方法,将肽段样品分成两份,对一份肽段使用甲醛进行N端二甲基化标记,使用H_(2)^(18)O进行C端^(18)O标记;对另一份样品使用氘代甲醛进行N端二甲基化标记,C端不做处理;两份样品等量混合后,相同序列的不同标记的肽段质量仅相差0.016 Da,这些准等重肽段可在质谱中同时碎裂,产生成对的碎片离子。通过发展基于碎片离子成对性的测序算法简化谱图,并有效提取和分辨b/y离子,实现对肽段快速从头测序。本方法对牛血清白蛋白酶解后肽段测序准确度达到95.51%,显著优于商品化测序软件PEAKS的测序准确度(57.96%)。同时,对单克隆抗体赫赛汀胰酶酶解后的肽段测序,测序准确度为93.60%。此方法可应用于对单克隆抗体序列表征等领域。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^(16)O of the C-terminal carboxyl group was replaced with^(18)O.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.

关 键 词：肽段测序 从头测序 准等重同位素标记 

分 类 号：O629.72[理学—有机化学]