Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography View Full Text


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Article Info

DATE

2013-02-07

AUTHORS

Houjiang Zhou, Mingliang Ye, Jing Dong, Eleonora Corradini, Alba Cristobal, Albert J R Heck, Hanfa Zou, Shabaz Mohammed

ABSTRACT

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 ∼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. More... »

PAGES

461-480

References to SciGraph publications

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  • 1983-05. Monodisperse polymer particles — a step forward for chromatography in NATURE
  • 2007-08-02. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips in NATURE PROTOCOLS
  • 2006-09-10. A probability-based approach for high-throughput protein phosphorylation analysis and site localization in NATURE BIOTECHNOLOGY
  • 2002-03. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae in NATURE BIOTECHNOLOGY
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  • 1994-11-01. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database in JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2013.010

    DOI

    http://dx.doi.org/10.1038/nprot.2013.010

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1043490961

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/23391890


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