title: Depletion of acidic phosphopeptides by SAX to improve the coverage for the detection of basophilic kinase substrates.
abstract: The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.
authors.affiliation: CAS Key Lab 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: Mingming
authors.initials: M
authors.lastname: Dong
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Chunxia
authors.initials: C
authors.lastname: Song
authors.forename: Yanbo
authors.initials: Y
authors.lastname: Pan
authors.forename: Chunli
authors.initials: C
authors.lastname: Wang
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000737:chemistry / Q000302:isolation & purification; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D016415:Sequence Alignment
chemical_list: D000838:Anions; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D010770:Phosphotransferases