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题名: Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography
作者: Zhou, Houjiang1, 2, 3;  Ye, Mingliang2;  Dong, Jing2;  Corradini, Eleonora1, 3;  Cristobal, Alba1, 3;  Heck, Albert J. R.1, 3;  Zou, Hanfa2;  Mohammed, Shabaz1, 3
刊名: NATURE PROTOCOLS
发表日期: 2013-03-01
DOI: 10.1038/nprot.2013.010
卷: 8, 期:3, 页:461-480
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods
研究领域[WOS]: Biochemistry & Molecular Biology
英文摘要: Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere-based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO2). Here we present a detailed protocol for the preparation of monodisperse microsphere-based Ti4+-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes similar to 2 weeks to complete the protocol: 1 week to prepare the Ti4+-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.
关键词[WOS]: MASS-SPECTROMETRIC ANALYSIS ;  COMPLEX PROTEIN MIXTURES ;  PHOSPHOPEPTIDE ENRICHMENT ;  SACCHAROMYCES-CEREVISIAE ;  PHOSPHORYLATED PEPTIDES ;  MS ANALYSIS ;  SITE LOCALIZATION ;  POLYMER PARTICLES ;  PROTEOMICS ;  TANDEM
语种: 英语
WOS记录号: WOS:000317110600003
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/137632
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Univ Utrecht, Utrecht Inst Pharmaceut Sci, Bijvoet Ctr Biomol Res, Utrecht, Netherlands
2.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian, Peoples R China
3.Netherlands Prote Ctr, Utrecht, Netherlands

Recommended Citation:
Zhou, Houjiang,Ye, Mingliang,Dong, Jing,et al. Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography[J]. NATURE PROTOCOLS,2013,8(3):461-480.
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