title: Selective Enrichment of Cysteine-Containing Phosphopeptides for Subphosphoproteome Analysis.
abstract: Among the natural amino acids, cysteine is unique since it can form a disulfide bond through oxidation and reduction of sulfhydryl and thus plays a pervasive role in modulation of proteins activities and structures. Crosstalk between phosphorylation and other post-translational modifications has become a recurrent theme in cell signaling regulation. However, the crosstalk between the phosphorylation and the formation and reductive cleavage of disulfide bond has not been investigated so far. To facilitate the study of this crosstalk, it is important to explore the subset of phosphoproteome where phosphorylations are occurred near to cysteine in the protein sequences. In this study, we developed a straightforward sequential enrichment method by combining the thiol affinity chromatography with the immobilized titanium ion affinity chromatography to selectively enrich cysteine-containing phosphopeptides. The high specificity and high sensitivity of this method were demonstrated by analyzing the samples of Jurkat cells. This "divide and conquer" strategy by specific analysis of a subphosphoproteome enables identification of more low abundant phosphosites than the conventional global phosphoproteome approach. Interestingly, amino acid residues surrounding the identified phosphosites were enriched with buried residues (L, V, A, C) while depleted with exposed residues (D, E, R, K). Also, the phosphosites identified by this approach showed a dramatic decrease in locating in disorder regions compared to that identified by conventional global phosphoproteome. Further analysis showed that more proline directed kinases and fewer acidophilic kinases were responsible for the phosphorylation sites of this subphosphoproteome.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , 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 Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Dong
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Keyun
authors.initials: K
authors.lastname: Wang
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , 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 Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D020543:Proteome; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D003545:Cysteine
keywords: crosstalk; cysteine-containing phosphopeptide; subphosphoproteome analysis