title: Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations.
abstract: Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Zheyi
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Mao
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China. Electronic address: wangfj@dicp.ac.cn.
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D020543:Proteome
keywords: Comparative phosphoproteome quantification; Extreme relative abundance; Mass spectrometry; Mixing at specific ratio; Stable isotope labeling