title: Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.
abstract: Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252.
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. 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; 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; 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.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000327:Adsorption; D000838:Anions; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D056945:Hep G2 Cells; D006801:Humans; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014025:Titanium
chemical_list: D000838:Anions; D010710:Phosphates; D010748:Phosphopeptides; D014025:Titanium
keywords: Competitive substitution; Mass spectrometry; Multi-phosphopeptide enrichment; Phosphoproteomics; Pre-coordination