title: Phosphoproteome profile of human liver Chang's cell based on 2-DE with fluorescence staining and MALDI-TOF/TOF-MS.
abstract: 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.
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Liu
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Zhuang
authors.initials: Z
authors.lastname: Lu
authors.forename: Liyan
authors.initials: L
authors.lastname: Jiao
authors.forename: Dongyang
authors.initials: D
authors.lastname: Zhang
authors.forename: Shaohui
authors.initials: S
authors.lastname: Sui
authors.forename: Ying
authors.initials: Y
authors.lastname: Jiang
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007526:Isoelectric Point; D008099:Liver / Q000166:cytology; D008110:Liver Extracts / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012394:Rosaniline Dyes; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D008110:Liver Extracts; D010750:Phosphoproteins; D020543:Proteome; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue