Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

Abstract

The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H 2O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode ® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.