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).

摘要

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 mul solvent of 0.4% TFA, 30.0% ACN and 69.6% H2O. 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 GelCodeReg. 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. All rights reserved, Elsevier.