Author:
Steen Hanno,Stensballe Allan,Jensen Ole N.
Abstract
INTRODUCTIONImmobilized metal ion affinity chromatography (IMAC) makes use of matrix-bound metals to affinity-purify phosphoproteins and phosphopeptides. Commonly used metals in early studies such as Ni2+, Co2+, Zn2+, and Mn2+ were shown to bind strongly to proteins with a high density of histidines. More recently, immobilized Fe3+, Ga3+, and Al3+ metal ions have been used for the selective enrichment of phosphopeptides from complex proteolytic digest mixtures containing both phosphorylated and nonphosphorylated components. The use of a nitrilotriacetic acid (NTA) matrix over iminodiacetic-acid-modified matrices has been reported to provide an advantage in selectivity. The development of elution conditions that are directly compatible with MS analysis of the enriched phosphopeptide samples provides the option to interface IMAC and MS online. This protocol describes the enrichment of phosphopeptides by IMAC using Fe3+- and Ga3+-NTA resin.
Publisher
Cold Spring Harbor Laboratory
Subject
General Biochemistry, Genetics and Molecular Biology
Cited by
4 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献