Electrostatic Repulsion Hydrophilic Interaction Chromatography — A New Tool for Enrichment of Phospho- and Glycopeptides

Tim Wehr

Posttranslationally modified sites in proteins are identified generally by digestion of the protein and identification of its constituent peptides. The low abundance of a particular posttranslationally modified species is a major impediment to its characterization in a complex mixture such as a body fluid or cell lysate. To obtain sufficient material for study and to eliminate the vast majority of interfering proteins, selective enrichment techniques are required. Enrichment can be performed at the protein level, or, more commonly, at the peptide level after enzymatic cleavage of the protein. Enrichment techniques for glycoproteins include lectin chromatography (1–3), size exclusion chromatography (4), hydrazide-based covalent coupling (5), and (in the case of sialic acid–containing glycopeptides) cation-exchange chromatography (6). Enrichment techniques for phosphoproteins and phosphopeptides were reviewed in the June issue of "Directions in Discovery" (7). These include immunoprecipitation (8,9), chemical modification by β-elimination (10) or using phosphoramidate chemistry (11), immobilized metal affinity chromatography (IMAC) (12), and enrichment on metal oxides such as titanium dioxide (13) or zirconium dioxide (14). Currently, IMAC or metal oxides are the most popular methods for enrichment of phosphopeptides.