Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 879, Issue 27, Pages 2852-2859Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2011.08.005
Keywords
Proteomics; Phosphoprotein; Phosphopeptide; Open tubular capillary; Polymer brush; Surface functionalization; IMAC enrichment
Funding
- European community
- Region Nord-Pas de Calais (France)
- IBISA network
- CNRS
- Universite de Lille 1, Sciences et technologies
- CNRS (France)
- CNRST (Morocco)
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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 alpha(S1), alpha(S2) mixture and beta 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 alpha(S1), alpha(S2) 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). (C) 2011 Elsevier B.V. All rights reserved.
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