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

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