Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry.

The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensitive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffer reduces nonspecific binding to the IMAC resin significantly, thereby improving the selectivity of IMAC for phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10-11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures.