Enhanced protein separation after removing soluble sugars from ground soybean seeds

Authors: ISLAM, NAZRUL - Oak Ridge Institute For Science And Education (ORISE); Luthria, Devanand - Dave; KOTHA, RAGHAVENDHAR - University Of Maryland; Natarajan, Savithiry - Savi

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2020
Publication Date: 8/29/2020
Citation: Islam, N., Luthria, D.L., Kotha, R.R., Natarajan, S.S. 2020. Enhanced protein separation after removing soluble sugars from ground soybean seeds. Analytical Biochemistry. 610:113931. https://doi.org/10.1016/j.ab.2020.113931.
DOI: https://doi.org/10.1016/j.ab.2020.113931

Interpretive Summary: Soybean is a rich source of oil, protein, carbohydrates, and other nutritional metabolites. But because of this complexity in seed composition, it is sometimes challenging to purifiy seed proteins from the other other seed components. It is thought that the removal of interfering substances can improve the extraction and subsequent analysis of seed proteins. In this study, ARS scientists developed an improved seed protein extraction method. The method involves two washing steps, one with hexane to reduce oil content and the second with alcohol to decrease soluble sugar interfering substances. This method improved protein separation and analysis of various proteins, specifically proteins of low abundance. The improved detection of proteins of low abundance will assist researchers in the federal government, at universities, or private institutions interested in peforming a deep analysis of the protein composition of soybean seeds.

Technical Abstract: Extraction and analysis of different soybean seed proteins are challenging because it contains a complex composition of seed storage proteins along with considerable amounts of fatty acids and soluble sugars. To characterize various proteins, it is essential to remove the other interfering components that might impact separation and analysis of different proteins. To remove the fat associated contaminants, the hexane washing technique is well adapted by the industries and research laboratories. Although alcohol leaching, a technique for removing soluble sugars, is routinely used in the industries, the efficacy of this technique and its influence on separation of different soy proteins under laboratory conditions has not been thoroughly investigated. Here, we demonstrate that hexane washing followed by alcohol leaching significantly reduced fatty acids and soluble sugar contents in soybean samples, thereby improving the resolution of various soybean proteins. We also developed and validated a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method to quantify the total amount of soluble sugars removed by alcohol leaching. Alcohol leaching removed about 60% of the total soluble sugars from the soybean seed powder, which, subsequently enhanced the protein separation using SDS-PAGE and 2-D-PAGE. The separated proteins were, isolated, trypsinized and identified 18 different proteins using Bruker amazon speed ion trap mass spectrometer equipped with ESI source.