Simplification of complex tryptic digests for capillary electrophoresis by affinity selection of histidine-containing peptides with immobilised metal ion affinity chromatography

Abstract

This paper reports on the selectivity behaviour of tryptic peptides on a Cu 2+-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI–TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10–75 m M SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 m M) was characterised by MALDI–TOF. When using SDS at 75 m M, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.