title: Preparation and loading buffer study of polyvinyl alcohol-based immobilized Ti4+ affinity chromatography for phosphopeptide enrichment.
abstract: Despite recent advances in phosphoproteomics, an efficient and simple enrichment protocol is still a challenge and of high demand aiming at large-scale plant phosphoproteomics studies. Here, we developed a novel loading buffer system for synthesized immobilized metal affinity chromatography material targeting plant samples, which was prepared by a simple one-step esterification between polyvinyl alcohol and phosphoric acid and then was subjected to immobilize Ti(4+). SEM and Fourier transform IR spectroscopy were used to assure the synthesis protocol of the polyvinyl alcohol-based Ti(4+) immobilized material, and the specific surface areas and pore volumes of the polymers were measured. The selectivity for phosphopeptide enrichment from α-casein was improved by optimizing the pH and components of the loading buffer. By using potassium hydrogen phthalate/hydrochloric acid with pH at 2.50 as the loading buffer, 19 phosphopeptides with high intensity were identified. The final optimized protocol was adapted to salt-stressed maize leaves for phosphoproteome analysis. A total of 57 phosphopeptides containing 59 phosphorylated sites from 50 phosphoproteins were identified in salt-stressed maize leaf. The research was meaningful to obtain much more information about phosphoproteins leading to the comprehension of salt resistance and salt-inducible phosphorylated processes of maize leaves.
authors.affiliation: Department of Environmental Sciences and Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing, P. R. China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Hu
authors.forename: Shuangxi
authors.initials: S
authors.lastname: Guo
authors.forename: Hongbo
authors.initials: H
authors.lastname: Ma
authors.forename: Ning
authors.initials: N
authors.lastname: Ye
authors.forename: Xueqin
authors.initials: X
authors.lastname: Ren
mesh_terms: D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009942:Organometallic Compounds / Q000737:chemistry*; D010748:Phosphopeptides / Q000302:isolation & purification*; D010756:Phosphoric Acids / Q000737:chemistry; D011142:Polyvinyl Alcohol / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013499:Surface Properties; D014025:Titanium / Q000737:chemistry*
chemical_list: D002021:Buffers; D009942:Organometallic Compounds; D010748:Phosphopeptides; D010756:Phosphoric Acids; D011142:Polyvinyl Alcohol; D014025:Titanium; C030242:phosphoric acid
keywords: Immobilized metal affinity chromatography; Maize; Phosphopeptide enrichment; Polyvinyl alcohol