title: Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison between polymer brush coating and surface functionalization.
abstract: In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe(3+), 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 α(S1), α(S2) 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 α(S1), α(S2) casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe(3+) 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).
authors: Université de Lille 1, Sciences et Technologies, USR CNRS 3290 Miniaturisation pour Synthèse, l'Analyse & Protéomique, 59655 Villeneuve d'Ascq, France.
authors: Khadija
authors: K
authors: El Idrissi
authors: Saïd
authors: S
authors: Eddarir
authors: Caroline
authors: C
authors: Tokarski
authors: Christian
authors: C
authors: Rolando
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D007098:Imino Acids / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D011109:Polymethacrylic Acids / Q000737:chemistry; D012821:Silanes / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013499:Surface Properties
chemical_list: C403136:3-glycidoxypropyltrimethoxysilane; D002364:Caseins; D007098:Imino Acids; D010748:Phosphopeptides; D011109:Polymethacrylic Acids; D012821:Silanes; C042535:polyglycidyl methacrylate; D007501:Iron; C008109:iminodiacetic acid