Phosphopeptide Purification by IMAC with Fe(III) and Ga(III)
This protocol was adapted from “Proteomic Methods for Phosphorylation Site Mapping,” Chapter 9, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Immobilized 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.
