Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests.

ABSTRACT: Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) g

SUBMITTER: Aryal UK 

PROVIDER: S-EPMC2628073 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature