|Hurkman Ii, William|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 4/1/2007
Publication Date: 6/1/2007
Citation: Vensel, W.H., Dupont, F.M., Chan, R., Hurkman, W.J. 2007. Mass Spectrometry Based Identifications of LMW Glutenin Subunits. In: George L. Lookhart, Perry K.W. Ng, editors. Gluten Proteins 2006. Minneapolis, MN: AACCI. P. 347-351. Interpretive Summary: Wheat storage proteins are important contributors to bread flour quality and are a major component of the human diet. Identification of specific proteins whose levels respond to environmental stimuli can aid in understanding the contribution of these proteins to wheat quality. Mass spectrometry has become an important tool to identify these proteins however some wheat storage proteins are difficult identify by this method. The proceedure reported here improves the reliability of mass spectrometry based identifications of wheat storage proteins that had been previously difficult to identify.
Technical Abstract: Tandem mass spectrometry (MS/MS) is routinely used to identify wheat endosperm proteins. In this method, peptide fragmentation patterns generated by MS/MS are identified using a ‘search engine’ to compare the spectra to those generated in silico from protein sequence databases. Trypsin is a commonly employed enzyme as it produces peptides that give good fragmentation patterns in the mass spectrometer. Gliadins and glutenins, however, are difficult to distinguish because they have a small number of cleavable tryptic sites and have very similar sequences, including repetitive motifs rich in proline and glutamine. Often glutenins and gliadins yield only one or two tryptic peptides that provide enough significant information for identification by MS/MS analysis. To address these problems we used enzymes with cleavage specificities different from trypsin. Preliminary results suggest that the number of peptides generated and detected is increased. All gel electrophoresis bands tested with trypsin gave identifiable fragments by electrospray ionization (ESI) MS/MS. Chymotrypsin yielded fewer fragments than trypsin while thermolysin produced fragments that allowed identification of the highly similar low molecular weight glutenin subunits (LMW-GS). This approach increased the number of peptides generated and detected. The improved MS analysis increased the confidence of MS/MS identifications of the glutenins and should similarly improve the identification of the gliadins.