Enhanced separation and analysis of low abundant soy proteins by dual washing extraction process

Soybean seeds provide a rich source of proteins, fats, carbohydrates, and micronutrients. Extraction and analysis of low abundant soybean seed proteins are challenging because of its complex seed composition. For characterizing various proteins, it is paramount to remove the other interfering components, primarily oils, and carbohydrates. In the present study, we used a sequential dual washing process initially with hexane to remove oil and non-polar interferences, followed by 80% ethanol washing to remove about 60% of the total soluble sugars. The extracted soluble sugars were quantified using a newly developed and validated high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This newly developed combined washings process significantly enhanced the separation of both low molecular weight and low abundant proteins using 1D (one dimensional)- and 2D (two dimensional) gel electrophoresis. The separated proteins were trypsinized and analyzed by using Bruker amazon speed ion trap mass spectrometer equipped with an ESI source. This combined washing process allowed the identification of 18 additional low abundant soy proteins as compared to the simple hexane washed samples. This purification process will allow researchers to identify and investigate the role of low molecular weight and low abundant proteins as it relates to plant functions, nutrition, and health.