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Improved identification of wheat prolamins through alkylation of cysteine residues and peptide-based mass spectrometry
I. ROMBOUTS (1), B. Lagrain (1), M. Brunnbauer (2), J. A. Delcour (1), P. Koehler (2). (1) KU Leuven, Leuven, Belgium; (2) TU Munich, Freising, Germany
Wheat flour quality greatly depends on the composition of its prolamins and the presence and location of cysteine residues in most of them. Wheat prolamin detection and quantification is also relevant in the context of celiac disease, especially since the commonly used ELISA technique often produces inconsistent results. LC-MS/MS, which has greatly advanced in recent years, is very useful for protein quantification and structure identification, <i>e.g.</i> to analyze the ratio of thiol and disulfide groups as affected by processing. In addition, isotope coded affinity tags (ICAT) for cysteine residues are increasingly used for quantitative proteomics. However, in the case of wheat prolamins, these techniques suffer from the low solubility of prolamins in buffers typically used for sample preparation. Here, methods are presented for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM), which significantly improve the detection of cysteine-containing peptides that can then be identified in enzymic prolamin digests by ESI-MS/MS. Optimal conditions for cysteine labeling, enzymic digestion of wheat prolamins, and detection of cysteine peptides are discussed. Finally, to the best of our knowledge, we were the first to label prolamins with a commonly used ICAT reagent. However, the extent of labeling was much higher with 4-VP and IDAM than with ICAT. The new protocol including alkylation with IDAM or 4-VP and LC-MS/MS of wheat prolamins offers perspectives for elucidation of the gluten structure and determination of gluten concentrations in (gluten-free) products.
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