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Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides
Published on: July 14, 2015
Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison
Khadija El Idrissi1, Saïd Eddarir, Caroline Tokarski
1Université de Lille 1, Sciences et Technologies, USR CNRS 3290 Miniaturisation pour Synthèse, l'Analyse & Protéomique, 59655 Villeneuve d'Ascq, France.
Summary
New open tubular columns using iminodiacetic acid (IDA) and iron(III) efficiently enrich phosphopeptides. Two chemistries, polymer brush coating and surface functionalization, show distinct selectivity for phosphorylated peptides.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Materials Science
Background:
- Phosphopeptide enrichment is crucial for proteomics.
- Existing methods often lack selectivity or efficiency.
- Immobilized Metal Affinity Chromatography (IMAC) is a common technique.
Purpose of the Study:
- To develop and compare two novel open tubular IMAC columns for phosphopeptide enrichment.
- To evaluate the performance of these columns using casein phosphopeptides.
- To investigate the selectivity of different surface chemistries for phosphopeptide enrichment.
Main Methods:
- Preparation of open tubular columns via in situ chemical modification of fused silica capillaries.
- Two chemistries were employed: poly-(glycidyl methacrylate) (GMA) brush and 3-glycidoxypropyltrimethoxysilane (GLYMO) surface functionalization.
- Immobilization of Fe(3+) onto iminodiacetic acid (IDA) functionalized surfaces.
- Optimization of sample loading, washing, and elution steps.
- Analysis of enriched phosphopeptides using MALDI-TOF mass spectrometry.
Main Results:
- Both GMA-IDA-Fe(3+) and GLYMO-IDA-Fe(3+) columns successfully enriched phosphopeptides from casein mixtures.
- Seven phosphorylated peptides, including a pentaphosphorylated peptide, were identified from the α(S1), α(S2) casein mixture.
- The GMA-IDA-Fe(3+) column eluted all phosphopeptides in a single fraction, irrespective of phosphorylation degree.
- The GLYMO-IDA-Fe(3+) column demonstrated selectivity, with longer elution times for peptides with higher phosphorylation states.
Conclusions:
- Open tubular IMAC columns functionalized with IDA-Fe(3+) offer efficient phosphopeptide enrichment.
- The choice of surface chemistry (GMA vs. GLYMO) influences the selectivity of phosphopeptide elution based on phosphorylation degree.
- These novel columns provide valuable tools for phosphoproteomics research.
