Lab-on-a-Chip method uncertanties in determination of high-molecular-weight glutenin subunits

Abstract

Polymeric wheat endosperm proteins, especially the high-molecular-weight glutenin subunits (HMW-GS), are probably the most interesting protein fraction giving the essential information about bread-making quality of wheat flour. A relatively new method that shows a great potential for a fast, reliable and automatable analysis of protein purity, sizing and quantification is microfluidic or Lab-on-a-Chip (LoaC) capillary electrophoresis. This work was aimed to explore the possibilities of implementation of LoaC method to analysis of protein samples isolated from a Serbian common wheat variety, emphasizing the steps that might bring uncertainties and affect reproducibility of obtained glutenin subunits quantitation results. A good resolution of protein bands in a molecular weight range of 14.0 to 220.0 kDa was achieved. The reproducibility of HMW-GS sizing and quantitation were good, with the average coefficient of variation values of 1.2% and 12.2%. The ratio of HMW-GS to low-molecular-weight glutenin subunits (LMW-GS) was about 20%. The investigation ruled out influences of the extract solution addition and the buffer addition steps of the applied method, as well as the individual chip influence on GS quantitation results. However, there was statistically significant difference between HMW-GS quantitation results of multi-step and one-step extraction procedures applied prior to glutenin subunits extraction step.