학술논문
Robust phosphoproteome enrichment using monodisperse microsphere--based immobilized titanium (IV) ion affinity chromatography
Document Type
Report
Author
Source
Nature Protocols. March 1, 2013, Vol. 8 Issue 3, p461, 20 p.
Subject
Language
English
ISSN
1754-2189
Abstract
INTRODUCTION Protein phosphorylation by kinases and dephosphorylation by phosphatases are key tools for signaling in cellular networks (1,2). Protein phosphorylation has been estimated to affect ~30% of a proteome and [...]
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 Ti[O.sub.2]). Here we present a detailed protocol for the preparation of monodisperse microsphere-based [Ti.sup.4+]-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 [Ti.sup.4+]-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.
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 Ti[O.sub.2]). Here we present a detailed protocol for the preparation of monodisperse microsphere-based [Ti.sup.4+]-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 [Ti.sup.4+]-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.