title: Pyridoxal 5'-phosphate mediated preparation of immobilized metal affinity material for highly selective and sensitive enrichment of phosphopeptides.
abstract: Phosphorylation is a crucial post-translational modification, which plays pivotal roles in various biological processes. Analysis of phosphopeptides by mass spectrometry (MS) is intractable on account of their low stoichiometry and the ion suppression from non-phosphopeptides. Thus, enrichment of phosphopeptides before MS analysis is indispensable. In this work, we employed pyridoxal 5'-phosphate (PLP), as an immobilized metal affinity chromatography (IMAC) ligand for the enrichment of phosphopeptides. PLP was grafted onto several substrates such as silica (SiO2), oxidized carbon nanotube (OCNT) and silica coated magnetic nanoparticles (Fe3O4@SiO2). Then the metal ions Fe3+, Ga3+and Ti4+were incorporated for the selective enrichment of phosphopeptides. It is indicated that Fe3O4@SiO2-PLP-Ti4+has a superior selectivity towards phosphopeptides under as much as 1000-fold interferences of non-phosphopeptides. Further, Fe3O4@SiO2-PLP-Ti4+exhibited high efficiency in selective enrichments of phosphopeptides from complex biological samples, including human serum and tryptic digested non-fat milk. Finally, Fe3O4@SiO2-PLP-Ti4+was successfully employed in the sample pretreatment for profiling phosphopeptides in a tryptic digest of rat brain proteins. Our experimental results evidenced a great potential of this new chelator-based material in phosphoproteomics study.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Xiao-Mei
authors.initials: XM
authors.lastname: He
authors.affiliation: Wuhan Institute of Biotechnology, Wuhan 430072, PR China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory of Tectonics and Petroleum Resources (Ministry of Education), China University of Geosciences, Wuhan 430075, PR China.
authors.forename: Gang-Tian
authors.initials: GT
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China; College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China.
authors.forename: Ren-Qi
authors.initials: RQ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China. Electronic address: yqfeng@whu.edu.cn.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Feng
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007477:Ions; D008280:Magnetics; D013058:Mass Spectrometry; D008670:Metals; D053758:Nanoparticles / Q000032:analysis; D010710:Phosphates / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D011730:Pyridoxal / Q000737:chemistry; D051381:Rats; D012822:Silicon Dioxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007477:Ions; D008670:Metals; D010710:Phosphates; D010748:Phosphopeptides; D011730:Pyridoxal; D012822:Silicon Dioxide
keywords: Immobilized metal affinity chromatography; Phosphopeptide enrichment; Pyridoxal 5′-phosphate; Rat brain