title: Phosphoproteome of crab-eating macaque cerebral cortex characterized through multidimensional reversed-phase liquid chromatography/mass spectrometry with tandem anion/cation exchange columns.
abstract: A fully automated online multidimensional liquid chromatography (MDLC) platform featuring high-/low-pH reversed-phase (RP) dimensions and two other complementary-strong anion exchange (SAX) and strong cation exchange (SCX), respectively-chromatographic separations in tandem, with conventional offline titanium dioxide pre-enrichment, has been applied for the first global phosphopeptide identification from the macaque cerebral cortex in the presence of phosphatase inhibitors. Phosphorylation data interpretation, including site determination, and network construction have been performed: 14,338 distinct phosphopeptides in 7572 non-redundant phosphosites at 1% FDR were identified with 784 novel phosphorylation sites when mapping into the two most-curated public phosphorylation databases, PhosphoSitePlus (PSP) and Phospho.ELM (ELM), using probability-based placements. The net charges of both extremely acidic and basic phosphopeptides depend largely on the pH of the solvent, in turn impacting their retention and subsequent fractionation; the inclusion of the complementary SAX and SCX column chemistries after the high-pH RP dimension allowed effective retention and separation of net-negatively and -positively charged phosphopeptides, thereby leading to extended anionic and cationic phosphopeptide coverage from basophilic and acidophilic kinase substrates. A valuable protein interaction network of known and predicted motifs kinases was constructed from 3064 confident phosphorylation sites in the non-human primate's brain.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Quan
authors.affiliation: Center for Bioinformatics and Computational Biology, and The Institute of Biomedical Sciences, School of Life Science, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
authors.forename: Jinwen
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Leong-Ting
authors.initials: LT
authors.lastname: Lui
authors.affiliation: Center for Bioinformatics and Computational Biology, and The Institute of Biomedical Sciences, School of Life Science, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
authors.forename: Tieliu
authors.initials: T
authors.lastname: Shi
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China. Electronic address: ivankchu@hku.hk.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D030562:Databases, Protein; D006863:Hydrogen-Ion Concentration; D008252:Macaca fascicularis / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D014357:Trypsin / Q000378:metabolism
chemical_list: D010748:Phosphopeptides; D014357:Trypsin
keywords: Macaca fascicularis; Mass spectrometry; Multidimensional liquid chromatography; Neuroproteomics; Phosphoproteome