期刊
JOURNAL OF SEPARATION SCIENCE
卷 36, 期 21-22, 页码 3563-3570出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.201300622
关键词
Immobilized metal affinity chromatography; Maize; Phosphopeptide enrichment; Polyvinyl alcohol
资金
- Chinese National Scientific Foundation [21175150, 21375146]
- Ministry of Education of China
- National Key Technology R & D Program, Ministry of Science & Technology of China [2011BAD11B02]
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 Ti4+. SEM and Fourier transform IR spectroscopy were used to assure the synthesis protocol of the polyvinyl alcohol-based Ti4+ immobilized material, and the specific surface areas and pore volumes of the polymers were measured. The selectivity for phosphopeptide enrichment from -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.
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