Quantitative reversed-phase high-performance liquid chromatographic analysis of wheat storage proteins as a potential quality prediction tool

Abstract

A selective precipitation procedure, in conjunction with reversed-phase high-performance liquid chromatography (RP—HPLC), was developed to facilitate quantitative analysis of high molecular weight (HMW) glutenin subunits. Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (gradient SDS—PAGE) and RP—HPLC analysis showed that the precipitation procedure provided a relatively clean separation of HMW glutenin subunits from lower molecular weight storage proteins, and individual alkylated HMW glutenin subunits were well-resolved by RP—HPLC, facilitating quantitative analysis. The procedure was tested for quantitative analysis of HMW glutenin subunits in a number of wheat cultivars and wheat backeross lines derived from Triticum speltoides with different HMW subunits and breadmaking quality characteristics. Storage proteins then were extracted sequentially with 50% propan-1-ol, 50% propan-1-ol containing 1 % dithiothreitol (DTT) and 50% propan-1-ol containing 4% DTT and 1 % acetic acid, and, in conjunction with selective precipitation and RP—HPLC, were analyzed quantitatively to determine the relative extractabilities of different protein groups, including the HMW subunits. Analysis of the good quality wheat Neepawa, which has HMW glutenin subunits 2 #x0002A; , 5, 7, 9 and 10, and two poor quality Neepawa backcross lines, with subunits 5 and 10 deleted, showed that the extractabilities of subunits 2 * , 7 and 9 increased significantly in the absence of subunits 5 and 10. Similar analysis of Manitou and a Manitou backcross line containing two novel subunits also suggested that relative extractability is affected by lower molecular weight proteins. The relative extractabilities of various storage protein fractions, as determined by RP—HPLC, was related inversely to quality data and, as such, may be useful for quality prediction.