Wheat cultivar‐specific proteins in grain revealed by 2‐DE and their application to cultivar identification of flour

Abstract

Wheat flour proteins were studied to identify the cultivar-specific proteins and use them to identify cultivars in flours. Proteins extracted from flours of Japanese wheat (cultivars Hokushin, Horoshirikomugi, Kitanokaori and Kachikei 33) and Canadian wheat (Canada Western Red Spring Wheat No. 1; 1CW) were analyzed by 2-DE with IEF gels over three pH ranges: pH 4-7, pH 5-8, and pH 6-11. This system enabled detection of more than 1600 protein spots. We recognized that among 50 protein spots showing cultivar-dependent qualitative changes, 25 proteins were wheat cultivar specific. These 50 protein spots were analyzed by N-terminal Edman degradation microsequencing and MALDI-TOF-MS; 21 protein spots were storage proteins, such as gliadin and low-molecular mass glutenin subunit. Five protein spots were identified as dehydroascorbate reductase (Triticum aestivum), triticin precursor (T. aestivum), alpha-amylase inhibitor (Oryza sativa), DNA-binding with one finger (Dof) zinc family protein (O. sativa), and nonphototropic hypocotyl 1 (NPH1) protein (Avena sativa). The other protein spots appeared to be hypothetical proteins (O. sativa or Arabidopsis thaliana) or functional unknown proteins. These specific proteins can be used as markers to identify wheat cultivars in blended flour composed of two or three flours.

Mass spectrometry analysis of gliadins in celiac disease

“…Of these, 21 were storage proteins, such as gliadin and low-molecular mass glutenin subunit. 51 Proteomic analysis of gliadins has to face a series of technical problems, which require a specifically dedicated approach. First, when the 70% ethanol-soluble gliadin fraction is used for separation on immobilised pH gradient (IPG) strips across the pH range 3-10, the most widely used in routine proteomic analysis, great part of the components migrate towards the alkaline side of the gel.…”

Comparative proteome analysis of seed storage and allergenic proteins among four narrow-leafed lupin cultivars

“…Very few preliminary studies using 2-DGE were reported on Lupinus angustifolius (Goggin et al, 2008;Sirtori et al, 2010) and there is no systematic proteomics-level study on this species. Since proteomic studies on grain species (Barakat, 2004;Kottapalli et al, 2008;Liu et al, 2009;Yahata et al, 2005) reported considerable variation of storage and allergenic proteins among cultivars, it is worth to study the important cultivars of narrow leafed lupin (NLL) to understand the expression level of different seed proteins. A fingerprinting study by our research group using direct mass spectrometry on 25 cultivars of NLL grown in Australia showed considerable seed protein variation among the cultivars.…”

Relationship between the Dough Quality and Content of Specific Glutenin Proteins in Wheat Mill Streams, and Its Application to Making Flour Suitable for Instant Chinese Noodles

“…1), making it very difficult to evaluate their correct amounts by SDS-PAGE. Thus, for a precise analysis of LMW-GS KS2, we investigated the amount of these proteins by using 2D-PAGE, as previously reported, 21) since this 2D- PAGE system enabled us to clearly isolate these proteins as nos. 6 and 7 in a mill stream (Fig.…”

“…The extra-strong flour-specific proteins of LMW-GS KS2 were analyzed according to the method described in the previous report 21) with a minor modification. The glutenin pellet was dissolved in 400 ml of a buffer containing 8 M urea, 2% (v/v) Triton X-100, 20 mM DTT and 2% (v/v) IPG (pH 6-11, Amersham Biosciences).…”

“…8) We have already established a proteomic analysis by 2D-PAGE, which enabled us to isolate more than 1,600 protein spots and reveal the qualitative changes among many kinds of wheat-cultivar flour. 21) This system made it possible to measure the amount of the glutenin protein, LMW-GS KS2, that had not previously been identified by SDS-PAGE. This information can be applied by millers and food manufacturers to establishing a milling system for producing high-quality mill streams with a high yield, and for blending extra-strong flour with soft flour to make good-quality flour suitable for instant Chinese noodles.…”