title: Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation.
abstract: Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.
authors.affiliation: German Research Center for Food Chemistry, Freising, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Lutz
authors.forename: Herbert
authors.initials: H
authors.lastname: Wieser
authors.forename: Peter
authors.initials: P
authors.lastname: Koehler
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D005433:Flour / Q000032:analysis; D005903:Gliadin / Q000737:chemistry; D005983:Glutens / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D014908:Triticum / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D005983:Glutens; D005903:Gliadin; C037302:glutenin