title: Improvement of the quantification accuracy and throughput for phosphoproteome analysis by a pseudo triplex stable isotope dimethyl labeling approach.
abstract: Accurately quantifying the changes of phosphorylation level on specific sites is crucial to understand the role of protein phosphorylation in physiological and pathological processes. Here, a pseudo triplex stable isotope dimethyl labeling approach was developed to improve the accuracy and the throughput of comprehensive quantitative phosphoproteome analyses. In this strategy, two identical samples are labeled with light and heavy isotopes, respectively, while another comparative sample is labeled with an intermediate isotope. Two replicated quantification results were achieved in just one experiment, and the relative standard deviation (RSD) criterion was used to control the quantification accuracy. Compared with the conventional duplex labeling approach, the number of quantified phosphopeptides increased nearly 50% and the experimental time was reduced by 50% under the same quantification accuracy. Combined with the automated online reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional separation system, a comparative phosphoproteome analysis of hepatocellular carcinoma (HCC) and normal human liver tissues was performed. Over 1800 phosphopeptides corresponding to ~2000 phosphorylation sites were quantified reliably in a 42 h multidimensional analysis. The pro-directed motifs, which were mainly associated with the extracellular signal-regulated kinases (ERKs), were observed as being overrepresented in the regulated phosphorylation sites, and some quantification results of phosphorylation sites were validated by the other studies. Therefore, this pseudo triplex labeling approach was demonstrated as a promising alternative for the comprehensive quantitative phosphoproteome analysis.
authors: CAS 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.
authors: Chunxia
authors: C
authors: Song
authors: Fangjun
authors: F
authors: Wang
authors: Mingliang
authors: M
authors: Ye
authors: Kai
authors: K
authors: Cheng
authors: Rui
authors: R
authors: Chen
authors: Jun
authors: J
authors: Zhu
authors: Yexiong
authors: Y
authors: Tan
authors: Hongyang
authors: H
authors: Wang
authors: Daniel
authors: D
authors: Figeys
authors: Hanfa
authors: H
authors: Zou
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D006801:Humans; D007553:Isotope Labeling; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D040901:Proteomics
chemical_list: D010748:Phosphopeptides