色谱

色谱 ›› 2019, Vol. 37 ›› Issue (5): 477-483.DOI: 10.3724/SP.J.1123.2018.11021

• 研究论文 • 上一篇    下一篇

基于超高效液相色谱-八端口转子阀的微升级预分级系统的建立

燕蒙1, 赵洋2, 张养军2, 应万涛2, 钱小红1,2   

  1. 1. 广东药科大学药学院, 广东 广州 510006;
    2. 军事科学院军事医学研究院生命组学研究所, 蛋白质组学国家重点实验室, 北京蛋白质组研究中心, 国家蛋白质科学中心-北京, 北京 102206
  • 收稿日期:2018-11-19 出版日期:2019-05-08 发布日期:2015-03-23
  • 通讯作者: 应万涛,Tel:(010)61777028,E-mail:yingwantao@mail.ncpsb.org;钱小红,Tel:(010)61777028,E-mail:qianxh1@163.com
  • 基金资助:

    国家重点研发计划（2017YFC0906600，2016YFA0501300）；国家自然基金重点项目（81530021）；军事医学创新工程专项（16CXZ027）.

Establishment of a microscale peptide prefractionation system based on ultra performance liquid chromatography and the eight-port rotor valve

YAN Meng1, ZHAO Yang2, ZHANG Yangjun2, YING Wantao2, QIAN Xiaohong1,2   

  1. 1. College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China;
    2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences(Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
  • Received:2018-11-19 Online:2019-05-08 Published:2015-03-23
  • Supported by:

    National Key Program for Basic Research of China (Nos. 2017YFC0906600, 2016YFA0501300); National Natural Science Foundation of China (No. 81530021); Innovation Project of Military Medicine (No. 16CXZ027).

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摘要：

复杂生物系统蛋白质组的深度覆盖鉴定得益于近年来快速发展的高效色谱分离和串联质谱分析技术。二维高效液相色谱（2-D HPLC）是实现复杂肽混合物正交分离的有效手段，但其缺点是运行时间长，通常要求肽上样量在毫克级别，且收集时的组分体积大，后续合并流程复杂，而有望替代其的StageTip技术，则由于有限的色谱分离梯度，难以达到充分的正交分离。该文探索了超高效液相色谱（UPLC）和八端口转子阀联用，作为高效、便捷肽段预分离和收集系统的可行性。研究结果显示，将UPLC的高pH反相色谱分馏与在线LC-MS/MS相结合，可以实现高pH和低pH条件下基于肽段不同色谱保留行为的正交互补分离，在蛋白质鉴定方面表现出优越的性能。应用该方法对人肝癌细胞系HepG2进行3次技术重复试验，重复试验间具有非常高的定量重现性（决定系数R2>0.95）。与传统StageTip方法相比，肽段鉴定数提高了23.52%。该分级方法灵活、稳定，能够针对较少的样品开展蛋白质组深度覆盖分析。

关键词: 八端口转子阀, 超高效液相色谱, 蛋白质组学, 二维液相色谱, 分馏

Abstract:

Liquid chromatography (LC) and mass spectrometry (MS)-based proteomics now allows very deep coverage of proteomes. Two-dimensional high performance liquid chromatography (2-D HPLC) is a useful tool for the proteome analysis of complex biosystem. However, it has drawback of a long running time, and it typically requires peptide amounts in the milligram range, and large volume of collected fractions. In this study, we introduce ultra-performance liquid chromatography (UPLC) and an eight-port rotor valve as a highly efficient and convenient method for a first-dimension separation and collection system. The combination of our UPLC-based fractionation using basic buffers with an online LC-MS/MS provided orthogonal peptide separation and demonstrated the powerful performance for protein identification. Upon applying the novel method to triplicate measurements of a human cell line, we observed excellent quantitative reproducibility between replicates (coefficient of determination R2>0.95) and more than 23.52% peptide identifications over the conventional StageTip approach. The fractionation method described here is flexible, straightforward, and robust, and it enables proteome analysis with minimal sample requirements.

Key words: fractionation, proteomics, the eight-port rotor valve, two-dimensional liquid chromatography (2-D LC), ultra performance liquid chromatography (UPLC)

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