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Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography
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
Source PublicationNATURE PROTOCOLS
2013-03-01
DOI10.1038/nprot.2013.010
Volume8Issue:3Pages:461-480
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
WOS SubjectBiochemical Research Methods
WOS Research AreaBiochemistry & Molecular Biology
WOS KeywordMASS-SPECTROMETRIC ANALYSIS ; COMPLEX PROTEIN MIXTURES ; PHOSPHOPEPTIDE ENRICHMENT ; SACCHAROMYCES-CEREVISIAE ; PHOSPHORYLATED PEPTIDES ; MS ANALYSIS ; SITE LOCALIZATION ; POLYMER PARTICLES ; PROTEOMICS ; TANDEM
AbstractMass 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.
Language英语
WOS IDWOS:000317110600003
Citation statistics
Cited Times:174[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/137632
Collection中国科学院大连化学物理研究所
Affiliation1.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
GB/T 7714
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.
APA Zhou, Houjiang.,Ye, Mingliang.,Dong, Jing.,Corradini, Eleonora.,Cristobal, Alba.,...&Mohammed, Shabaz.(2013).Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography.NATURE PROTOCOLS,8(3),461-480.
MLA Zhou, Houjiang,et al."Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography".NATURE PROTOCOLS 8.3(2013):461-480.
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