色谱

色谱 ›› 2014, Vol. 32 ›› Issue (4): 349-354.DOI: 10.3724/SP.J.1123.2013.10030

• 庆祝《色谱》创刊三十周年暨卢佩章院士九十华诞专刊-研究论文 • 上一篇    下一篇

利用谱图校正提高色谱-质谱联用的分析效率

陆文渊1,2, 张扬2, 沈诚频2, 殷薛飞2, 刘晓慧2, 杨芃原1   

  1. 1. 复旦大学化学系, 上海 200433;
    2. 复旦大学上海医学院生物医学研究院, 上海 200032
  • 收稿日期:2013-10-29 修回日期:2013-11-28 出版日期:2014-04-08 发布日期:2014-03-28
  • 通讯作者: 杨芃原
  • 基金资助:

    国家重大科学计划项目（2010CB912700，2013CB911201，2011CB910600）；国家高技术研究发展计划项目（2012AA020203，2014AAQ00361）；国家自然科学基金重点项目（21227805）.

A method using spectrum alignment to improve analysis efficiency of liquid chromatography combined with mass spectrometry

LU Wenyuan1,2, ZHANG Yang2, SHEN Chengpin2, YIN Xuefei2, LIU Xiaohui2, YANG Pengyuan1   

  1. 1. Department of Chemistry, Fudan University, Shanghai 200433, China;
    2. Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
  • Received:2013-10-29 Revised:2013-11-28 Online:2014-04-08 Published:2014-03-28

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摘要：

在使用对二甲基亚砜（DMSO）和NEDD8激活酶抑制剂（MLN4924，MLN）刺激的人脐静脉内皮细胞（HUVEC）内的蛋白质进行分析的过程中，利用Progenesis LC-MS软件对色谱图进行了保留时间的校正，并比较了同组分多次重复实验中的谱图相似率及两种刺激下细胞内蛋白质色谱图的相似度。样品经双酶切处理后，加入QconCAT标准蛋白质混合物作为参照，经高效液相色谱-串级质谱分离，后续又对谱图进行了校正与分析。经过谱图校正，将蛋白质鉴定结果从7000个左右提高到8000个以上，提高了蛋白质的鉴定效率。在利用谱图计数进行相对定量时，还分析了DMSO和MLN分别刺激HUVEC后细胞内的蛋白质差异在1000个左右，并给出了校正后的色谱总离子流图的相似度比较。相比其他方法更为简单快捷和流程化，具有高通量高灵敏度的优点。

关键词: 蛋白质分析, 高效液相色谱-串级质谱, 谱图校正

Abstract:

In the analysis of proteins in human umbilical vein endothelial cells (HUVEC) treated with dimethyl sulfoxide (DMSO) and NEDD8-activating enzyme inhibitor (MLN4924,MLN), the Progenesis LC-MS software (Nonlinear Dynamics Ltd) was applied to liquid chromatography spectrum alignment, while spectrum similarities were figured out among several experiments of the same sample, and also among different samples. After double enzymolysis, the sample was added with digested QconCAT standard proteins. They were separated by HPLC-MS/MS, followed by spectrum alignment and data analysis. This established experiment flow offered a better identification result of more than 8000 proteins, while the original result was about 7000 proteins, ensuring a relatively high identification efficiency. On the basis of relative quantification with spectrum count, the described procedure can analyze the differential expression of proteins induced by DMSO and MLN. The similarities of total ion chromatograms after alignment were also compared. This method was proved to be quick and easy, with the advantages of high throughput and high sensitivity.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), protein analysis, spectrum alignment

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