Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: Comparison between polymer brush coating and surface functionalization

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe3+, were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an αS1, αS2 mixture and β casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the αS1, αS2 casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe3+ IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).