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J Proteomics. 2014 Jan 16;96:253-62. doi: 10.1016/j.jprot.2013.11.014. Epub 2013 Nov 22.

An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.

Author information

1
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
2
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
3
The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, China.
4
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: mingliang@dicp.ac.cn.
5
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: hanfazou@dicp.ac.cn.

Abstract

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.

KEYWORDS:

Glu-C digestion; Human liver phosphoproteome; LTQ Orbitrap Velos; Mass spectrometer; RP-RPLC; TripleTOF 5600

PMID:
24275569
DOI:
10.1016/j.jprot.2013.11.014
[Indexed for MEDLINE]

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