Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Publications at this Location » Publication #236541

Title: Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

item Vensel, William
item Altenbach, Susan
item Dupont, Frances
item Tanaka, Charlene
item Hurkman Ii, William

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/12/2009
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The fragmentation information is searched against the predicted fragmentation pattern of peptides generated from an in silico protein sequence database. Contrary to expectations, only a small number of the likely peptides are observed. Factors contributing to this include: incomplete enzymatic cleavage, few cleavage sites for the chosen enzyme, ion suppression in the mass spectrometer, composition of the database, and the search engine(s) used. Particularly challenging are proteins that are members of highly similar families containing repetitive sequence motifs and proteins with few tryptic cleavage sites. The wheat gluten proteins display these characteristics. The gluten proteins are of great commercial value and play a major role in human nutrition because their viscoelastic properties lend them to the production of a wide variety of food products. They are composed of the monomeric gliadins (&alpha, &gamma and &omega types) and polymeric glutenins. The glutenins are composed principally of the low molecular weight glutenin subunits (LMW-GS) and high molecular weight glutenin subunits (HMW-GS). Homologs of these proteins originate from the three genomes of hexaploid bread wheat, whose family members are similar and have repetitive protein sequences with an abundance of glutamine and proline. To increase MS/MS protein sequence coverage of the wheat gluten proteins, we used several enzymatic cleavages (chymotrypsin and thermolysin in addition to trypsin), constructed a database containing wheat sequences derived from more four different wheat EST assemblies, analyzed spectral data with multiple search engines and used a commercially available software package (Scaffold) to combine and visualize all results. The fewest peptide fragments and identified proteins were obtained with trypsin. The effectiveness of the different enzymes varied depending upon the protein type fragmented. Chymotrypsin was more effective than thermolysin in cleaving alpha, gamma and omega gliadin proteins. However, thermolysin produced a greater number of peptides for the LMW-GS than did chymotrypsin or trypsin, while the HMW-GS were effectively cleaved by both thermolysin and trypsin. Database searching with X!Tandem and Mascot version 2.1 found 41 unique proteins using chymotrypsin alone, 45 with thermolysin and with trypsin 25. The effectiveness the different enzymes varied depending upon the protein type fragmented. Thermolysin produced a greater number of peptides of the LMW-GS than did chymotrypsin or trypsin. However the HMW-GS were effectively cleaved by both thermolysin and by trypsin. Chymotrypsin cleavage often resulted in protein identifications with more peptide fragments than either of the other two enzymes. However thermolysin was much more effective than trypsin in producing protein identifications and a large number of fragments per protein. Combining the search results for all enzymes and search engines increased the overall sequence coverage and the number of proteins identified to 56.