Proteomic‐based analytical approach for the characterization of glutenin subunits in durum wheat

Abstract

One of the main objectives of wheat glutenin subunit (GS) analysis is the identification of protein components linked to wheat quality. The proteomic characterization of glutenin has to consider the relatively low levels of arginine and lysine residues and the close sequence similarity among the different groups of these subunits, which hinders or even prevents the identification of the GS. In this study, a proteomic approach has been applied to resolve the heterogeneity of wheat glutenin components. Proteins extracted from Triticum durum flour were first analyzed by two-dimensional gel electrophoresis, which greatly reduced glutenin complexity. The identity of each spot was confirmed by nano liquid chromatography tandem mass spectrometry analysis of tryptic peptides. In parallel, measurements of the high mass range by matrix-assisted laser desorption/ionization time-of-flight analysis allowed detection of the large tryptic peptides. Gathering all data from search engine interrogation, very high sequence coverage was obtained for high molecular weight GS, including Bx7 and By8, in agreement with the known genetic profile of durum wheat. In addition, a truncated form of By8, never detected before, was also found. Low molecular weight GS (LMW-GS) B-type was identified with reasonable sequence coverage, while a clear identification of LWM-GS C- and D-type was hindered by the incompleteness of the wheat DNA databases. This study represents the first comprehensive analysis of the glutenin proteome and provides a reliable method for classifying wheat varieties according to their glutenin profile.

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

“…A number of factors should be considered when interpreting proteomic studies of the wheat grain proteins. Several studies have shown that the same protein sequence may be associated with multiple 2-DE spots with different pIs and molecular masses Gao et al, 2009;Mamone et al, 2009;Yang et al, 2011). Indeed, 32 proteins in Table 2 were identified in multiple spots in one or more of the studies.…”

“…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.…”

One Hundred Years of Grain Omics: Identifying the Glutens That Feed the World

“…Although mass spectra can potentially differentiate between wheat varieties, the unequivocal identification of LMW and gliadin subunits in unfractionated extracts through direct MALDI-TOF-MS analysis as a stand-alone technique, may be precluded by the pattern complexity 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 The use of peptide sequence data from tandem mass spectrometry (MS/MS) measurements, known as peptide fragment fingerprint (PFF), can be used to interrogate search engines to look for parent proteins 75 . However, the identification of gluten proteins by this approach is currently hindered by the limited information contained in gene and protein databases.…”

Analysis of gluten proteins composition during grain filling in two durum wheat cultivars submitted to two water regimes

“…In agreement with Shewry and Halford (2002) Concerning the ratio volume between the two phenological stages, a different behavior was observed for the different gel regions. As for H region, in the literature it is reported that the two cultivars under study show the presence of Glu-B1 b allele coding for protein subunits HMW-GS 7 and 8 associated with favorable dough properties (Gianibelli et al, 2001;Giuliani et al, 2012;Mamone et al, 2009;Yan et al, 2009). In the control treatment the increase in protein expression observed at physiological stage was more marked in Ciccio (Table 1).…”