色谱

色谱 ›› 2013, Vol. 31 ›› Issue (10): 927-933.DOI: 10.3724/SP.J.1123.2013.04041

• 研究论文 • 上一篇    下一篇

酶切过程中肽段过烷基化对蛋白质定性和定量分析的影响

王继峰1,2, 赵新元1,2, 赵焱2, 马成2, 钟儒刚1, 钱小红1,2, 应万涛2   

  1. 1. 北京工业大学生命科学与生物工程学院, 北京 100022;
    2. 蛋白质组学国家重点实验室, 北京蛋白质组研究中心, 北京放射医学研究所, 北京 102206
  • 收稿日期:2013-04-23 修回日期:2013-05-13 出版日期:2013-10-28 发布日期:2013-10-08
  • 通讯作者: 应万涛
  • 基金资助:

    国家重点基础研究"973"项目(2012CB910301);国家高技术研究发展计划项目(2012AA020201);国家自然科学基金项目(31100591).

Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins

WANG Jifeng1,2, ZHAO Xinyuan1,2, ZHAO Yan2, MA Cheng2, ZHONG Rugang1, QIAN Xiaohong1,2, YING Wantao2   

  1. 1. College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China;
    2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China
  • Received:2013-04-23 Revised:2013-05-13 Online:2013-10-28 Published:2013-10-08

PDF

580

被引次数

5

摘要：

蛋白质的还原-烷基化是蛋白质酶切中的重要步骤,常用的烷基化试剂是碘乙酰胺(IAA),但是IAA除了和半胱氨酸发生反应,也可能和其他多种氨基酸发生副反应。我们模拟常规的酶切条件,系统地研究了蛋白质真实酶切时所有酶切肽段发生烷基化的情况。结果表明,多种氨基酸可以发生烷基化,其趋势为:半胱氨酸>肽段N端氨基酸>天冬氨酸>谷氨酸>组氨酸>天冬酰胺>赖氨酸>酪氨酸,同时也发现同一肽段上的氨基酸烷基化具有排他性和聚集性。根据定性结果,采用质谱多反应监测(MRM)技术对多个肽段进行了定量分析,评估了过烷基化对蛋白质定量分析的影响。该研究结果表明,过量的烷基化修饰对蛋白质的定性与定量分析都可能产生较大影响。在蛋白质组学研究的样本处理流程中,应避免样本的过烷基化。

关键词: 蛋白质组学, 碘乙酰胺, 多反应监测, 烷基化, 液相色谱, 质谱

Abstract:

Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine>N-terminal amino acid>aspartic acid>glutamic acid>histidine>asparagine>lysine>tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.

Key words: alkylation, iodoacetamide, liquid chromatography (LC), mass spectrometry (MS), multiple reaction monitoring (MRM), proteomics

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