An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome
An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome
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用于深入分析人肝脏磷酸蛋白质组的酶辅助 RP-RPLC 方法
DOI：
10.1016/j.jprot.2013.11.014
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发表时间：
2014-01-16
影响因子：
3.3
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Bian, Yangyang;Song, Chunxia;Zou, Hanfa
蛋白质磷酸化是最常见的翻译后修饰之一。它在调节肝组织的多种生物学过程中起着关键作用。为了更好地了解蛋白磷酸化在肝脏功能中的作用，有必要对人类肝脏进行深入的磷酸化蛋白质组分析。在这里，酶辅助反相-反相液相色谱（RP-RPLC）的方法与两个RPLC分离操作与优化的酸性移动的相开发。通过胰蛋白酶消化从第一次RPLC分离收集的级分中的Glu-C产生的肽，实现了高度正交分离。通过使用两种类型的仪器，即TripleTOF 5600和LTQ Orbitrap Velos，磷酸化蛋白质组覆盖率进一步提高。最终从正常人肝组织中鉴定出22，446个磷酸化位点，对应于6526个非冗余磷蛋白。在这些位点中，15，229个位点被确信地定位为Ascore >= 13。该数据集是人类肝脏最大的磷酸化蛋白质组数据集。它可以是一个公共资源的肝脏研究界和持有的承诺，为进一步的生物学studies.Biological significance酶辅助的方法，使两个RPLC分离操作都与优化的酸性移动的相。TripleTOF 5600和Orbitrap Velos的鉴定具有高度互补性。生成了人类肝脏最大的磷酸化蛋白质组数据集。(C)2013爱思唯尔有限公司版权所有。
Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore >= 13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies.Biological significance The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated. (C) 2013 Elsevier B.V. All rights reserved.