4.5 Article

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

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ELSEVIER
DOI: 10.1016/j.jchromb.2011.08.005

关键词

Proteomics; Phosphoprotein; Phosphopeptide; Open tubular capillary; Polymer brush; Surface functionalization; IMAC enrichment

资金

  1. European community
  2. Region Nord-Pas de Calais (France)
  3. IBISA network
  4. CNRS
  5. Universite de Lille 1, Sciences et technologies
  6. CNRS (France)
  7. 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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