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Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS
Author(s) -
Liu Jinfeng,
Cai Yun,
Wang Jinglan,
Zhou Qi,
Yang Bing,
Lu Zhuang,
Jiao Liyan,
Zhang Dongyang,
Sui Shaohui,
Jiang Ying,
Ying Wantao,
Qian Xiaohong
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600696
Subject(s) - phosphoprotein , phosphorylation , coomassie brilliant blue , staining , protein phosphorylation , microbiology and biotechnology , biology , chemistry , biochemistry , protein kinase a , genetics
Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro‐Q Diamond dye, for the high‐throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2‐DE, then sequentially stained with Pro‐Q Diamond and Coomassie Blue G‐250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI‐TOF/TOF‐MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2‐DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection.