Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 3, 2011
Publication Date: February 1, 2011
Citation: Vensel, W.H., Dupont, F.M., Altenbach, S.B., Sloane, S.M. 2011. Effect of Cleavage Enzyme, Search Algorithm and Decoy Database on Mass Spectrometric Identification of Wheat Gluten Proteins. Phytochemistry. 72(10):1154-1161. Interpretive Summary: Wheat storage proteins are major determinants of bread quality, play a central role in human nutrition and are of great economic value. Broadly speaking there are four major classes of the storage proteins. Proteins within a given class are very similar in structure and difficult to distinguish. Some proteins are associated with human allergies and others with celiac disease. Under different environmental conditions there is a change in the amount of various proteins within a class. These changes are associated with changes in bread quality. It is important to be able to differentiate among the proteins in each class because proteins with similar sequences can have different effects on functional properties of flour and on the promotion of celiac disease. The methodology described in this paper improves and standardizes methods for distinguishing among these very similar proteins by using mass spectrometry.
Technical Abstract: Tandem mass spectrometry (MS/MS) is routinely used to identify proteins by comparing peptide spectra to those generated in silico from protein sequence databases. Wheat storage proteins (gliadins and glutenins) are difficult to distinguish by MS/MS as they have few cleavable tryptic sites, often resulting in zero to two tryptic peptides that provide insufficient information for identification. Wheat has a very large partially sequenced genome, the sequence information is scattered among a number of databases, there are less than 13,000 complete protein sequences available and the sequences are similar and have repetitive motifs. Thus protein databases were constructed that included sequences derived from several wheat EST assemblies. In addition, gluten protein sequences were translated from DNA sequences assembled from ESTs from the cultivar under study and added to the databases. We examined the effect of protein database content and size on the false discovery rate. To increase the number of peptides released by enzymatic cleavage we used chymotrypsin and thermolysin as well as trypsin to fragment the proteins. MS parameters were adjusted to improve detection of chymotryptic and thermolytic peptides. Since the software used to search the database can influence the results we employed two different search engines. When these data were combined and analyzed it was found that the identified peptides and proteins increased.