Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

Abstract

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.

Immune Cross-Reactivity in Celiac Disease: Anti-Gliadin Antibodies Bind to Neuronal Synapsin I

“…The patient data also clearly demonstrated that antigliadin Ab levels do not necessarily correlate with anti-synapsin Ab reactivity and that only certain subsets of anti-gliadin Abs cross-react with synapsin I. Because of the large number and heterogeneous nature of gliadins, as well as the high diversity of wheat phenotypes (26), the anti-gliadin immune response is likely to involve a sizeable repertoire of antigenic determinants. Therefore, varying degrees of cross-reactivity to synapsin I can be expected in different patients with gluten sensitivity.…”

New insights into the effects of high temperature, drought and post-anthesis fertilizer on wheat grain development

“…The proteins have highly repetitive sequences with an abundance of glutamine and proline and yield few peptides when cleaved with trypsin, the enzyme used in most MS studies. Additionally, current sequence databases do not reflect the considerable heterogeneity that is found among individual proteins in different cultivars (Altenbach et al, 2010a,b;Mamone et al, 2005Mamone et al, , 2009. Recently, Vensel et al (2011) optimized MS/MS methods for identification of gluten proteins by digesting each protein with chymotrypsin and thermolysin in addition to trypsin, using improved search strategies for analysis of spectral data, and including cultivar-specific sequences of gluten proteins in databases searched with spectral data.…”

A reply to Donald D. Kasarda: Lack of intestinal mucosal toxicity ofTriticum monococcumin celiac disease patients

“…We agree with Dr. Kasarda that further work is needed to determine the identity of gliadins and their potential toxicity, and we are currently characterizing T. monococcum gliadins, using a proteomicsbased approach [11].…”