Extraction of up to 95% of Wheat (Triticum aestivum) Flour Protein Using Warm Sodium Dodecyl Sulfate (SDS) without Reduction or Sonication

Abstract

Extraction of glutenin polymers without sonication is an essential prerequisite for accurate determination of their composition and molecular size distribution. Sequential fractionation of wheat flour with 0.1 M KCl and 0.25% sodium dodecyl sulfate (SDS) at 21 degrees C and 2% SDS at 60 degrees C extracted up to 95% of total protein. We propose that 2% SDS at 60 degrees C disrupts hydrogen bonds in glutenin and gliadin aggregates, reduces hydrophobic interactions, and facilitates solubilization. Analysis by size-exclusion high-performance liquid chromatography (SE-HPLC), reverse-phase (RP)-HPLC, and SDS-polyacrylamide gel electrophoresis (PAGE) revealed that partitioning of gliadins and glutenins among the extracts differed for two flours with good baking quality (Butte 86 and Jagger) and one with poor baking quality (Chinese Spring). More gliadin was associated with the 0.25% SDS extract for Chinese Spring, whereas more gliadin was associated with the 2% SDS extract for Butte 86 and Jagger. Unextractable glutenin polymer was only 4-5% of total protein for Butte 86 and Chinese Spring and 14% for Jagger.

Physicochemical control of durum wheat grain filling and glutenin polymer assembly under different temperature regimes

“…Extracts were fractionated by sizeexclusion high-performance liquid chromatography (SE-HPLC) on a TSK G4000SWXL column (Tosoh Europe N.V., Lyon, France) using phosphate elution buffer including 0.1% (w/v) SDS with detection at 214 nm. Chromatograms of SDS-soluble extracts were integrated into five fractions (large glutenin polymers, 750,000 < Mr < 2,000,000; small glutenin polymers, 90,000 < Mr < 750,000; u-gliadins 55,000 < Mr < 90,000; g-/bgliadins, 18,000 < Mr < 55,000; albumin/globulin and a-gliadins, 5000 < Mr < 18,000) on the basis of elution time and according to the known molecular size range of the different soluble wheat storage proteins (Dupont et al, 2008). Results are presented as mg of N per grain, percent of glutenin polymers or as percent of total protein, calculated from the sums of the areas of the chromatograms of SDS-insoluble and SDS-soluble fractions, after correction for solid to solvent ratios.…”

Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

“…Proteins were extracted from milled flour with 2% SDS by the method of (DuPont et al, 2008). Briefly, albumins and globulins were extracted from 400 mg of flour with 100 mM KCl and 5 mM EDTA in 50 mM Tris-HCl, pH 7.8.…”

A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat

“…Extracts were analyzed by SDS-PAGE, immunoblotting and RIDASCREEN ® Gliadin competitive ELISA. The remaining proteins (mostly albumins and globulins) in the residue were extracted with 25 mM Tris-HCl (pH 8.0) containing 2% SDS (1:20, w/v) and finally with 25 mM Tris-HCl (pH 8.0) containing 2% (w/v) SDS and 1% (w/v) DTT (1:20, w/v) [35]. Both for 30 min at 60 • C with mixing every 5-10 min, followed by centrifugation at 14,000 rpm for 10 min at room temperature.…”