Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns.

ABSTRACT: Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO(2), Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer

SUBMITTER: Ruprecht B 

PROVIDER: S-EPMC4288255 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature