Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate‐based buffers

Abstract

This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.

Characterization and Comparative Analysis of Wheat High Molecular Weight Glutenin Subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS

“…The HMW-GS elution order under acidic buffer CE condition was 1Dy f 1By f 1Bx f 1Ax f 1Dx. As indicated by previous investigations (13,14,35), the peaks of individual subunits displayed isoforms under acidic separation condition and similar results were obtained by two-dimensional gel electrophoresis (data not shown), which probably resulted from sample treatment or post-translational modifications (PTMs). In most cases, each subunit had one major peak and one or two minor peaks.…”

“…A BioFocus-3000 instrument was used for capillary electrophoretic separations of the HMW-GS according to the method of Yan et al (13) with minor modification in the analysis time. Acidic CE buffer, namely, 0.1 M phosphate-glycine (pH 2.5), containing 20% (v/v) acetonitrile (ACN) and 0.05% (w/v) hydroxypropylmethylcellulose (HPMC), and uncoated fused-silica capillaries (BioFocus-3000 accessory) of 25.5 cm length (20 cm to detector) and 50 μm inner diameter (i.d.)…”

“…Therefore, HMW-GS have become useful protein markers for wheat quality improvement and cultivar identification. In the past several decades, considerable efforts have been focused on developing powerful techniques for fast differentiation of good-and poor-quality HMW-GS in wheat for germplasm screening and quality improvement (11)(12)(13)(14)(15). Up to now, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the most popular method to detect the allelic compositions of HMW-GS on the basis of their mobilities (16).…”

Capillary electrophoresis of proteins 2003–2005

“…For example, CZE has been used for identification of glutenin subunits from different hexaploid wheat species [222,226]. Lines belonging to the same species can be discriminated mainly on the basis of b-and o-glutenin patterns, while g-and ogliadins seem to be more suitable for differentiation of emmer (durum wheat) from spelt (common wheat) [229].…”

“…Analysis of proteins in cereal cultivars has widely shown the importance of CE of cereal proteins for quality control [220][221][222][223][224][225][226][227][228]. Alcohol extracts from wheat grain typically contain gliadins, whereas water extracts contain mainly albumins.…”