Yan Meng, Zhao Yang, Zhang Yangjun, Ying Wantao, Qian Xiaohong
College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences(Beijing), Beijing Institute of Lifeomics, Beijing 102206, China.
Se Pu. 2019 May 8;37(5):477-483. doi: 10.3724/SP.J.1123.2018.11021.
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 >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.
基于液相色谱（LC）和质谱（MS）的蛋白质组学现在能够对蛋白质组进行非常深入的覆盖。二维高效液相色谱（2-D HPLC）是分析复杂生物系统蛋白质组的一种有用工具。然而，它存在运行时间长的缺点，通常需要毫克级别的肽量以及大量收集的馏分。在本研究中，我们引入超高效液相色谱（UPLC）和八通旋转阀，作为一种用于一维分离和收集系统的高效便捷方法。我们基于使用碱性缓冲液的UPLC分级分离与在线LC-MS/MS相结合，实现了正交肽分离，并证明了其在蛋白质鉴定方面的强大性能。将这种新方法应用于人类细胞系的三次重复测量时，我们观察到重复测量之间具有出色的定量重现性（决定系数>0.95），并且与传统的StageTip方法相比，肽鉴定数量增加了超过23.52%。这里描述的分级分离方法灵活、直接且稳健，能够以最少的样品需求进行蛋白质组分析。
