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Characterization of wheat gliadin proteins by combined two‐dimensional gel electrophoresis and tandem mass spectrometry
Author(s) -
Mamone Gianfranco,
Addeo Francesco,
Chianese Lina,
Di Luccia Aldo,
De Martino Alessandra,
Nappo Annunziata,
Formisano Annarita,
De Vivo Pasqualina,
Ferranti Pasquale
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401168
Subject(s) - gliadin , glutenin , chemistry , bottom up proteomics , protein mass spectrometry , tandem mass spectrometry , mass spectrometry , chromatography , isoelectric focusing , peptide mass fingerprinting , electrospray ionization , gel electrophoresis , proteome , peptide sequence , isobaric labeling , isoelectric point , proteomics , biochemistry , protein subunit , gluten , enzyme , gene
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 α/β‐, γ‐, ω‐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. α‐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.