Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides

A novel procedure for micropurification of phosphorylated peptides, as a front end to mass spectrometric analysis, is described. As a result of a systematic study, we propose the use of an immobilized metal affinity chromatography (IMAC) in a microtip (Erdjument-Bromage, H.; et al. J. Chromatogr., A 1998, 826, 167-181) format, more specifically in combination with Ga(III) ions. Manual Ga-(III) IMAC is easy to perform; phosphopeptides are retrieved in a near-quantitative and highly selective manner, to yield a concentrated sample for direct analysis by matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization mass spectrometry. Ga-(III) ions offer distinct advantages over the use of other metals, such as Fe(III) and Al(III), in terms of both selectivity and versatility, including facile base elution. Selectivity is best illustrated by effective enrichment of phosphopeptides that were present in a molar ratio of approximately 2% on a background of unphosphorylated protein, a situation very typical perhaps for protein phosphorylation states in the cell. The system was also used to retrieve and tentatively identify five previously uncharacterized phosphopeptides from a tryptic digest of human beta 4 integrin, isolated from cell extracts by immunoprecipitation.