Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategy.
The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.
Sui
Shaohui
S
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, People's Republic of China.
Wang
Jinglan
J
Yang
Bing
B
Song
Lina
L
Zhang
Jiyang
J
Chen
Ming
M
Liu
Jinfeng
J
Lu
Zhuang
Z
Cai
Yun
Y
Chen
Shuo
S
Bi
Wei
W
Zhu
Yunping
Y
He
Fuchu
F
Qian
Xiaohong
X
D001665:Binding Sites; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
D010750:Phosphoproteins