Proteomic comparison of three extraction methods reveals the abundance of protease inhibitors in the seeds of grass pea, a unique orphan legume

Authors: Miranda, Carrie; XU, QUANLE - Northwest A&f University; Oehrle, Nathan; ISLAM, NAZRUL - Orise Fellow; Garrett, Wesley; Natarajan, Savithiry - Savi; Gillman, Jason; Krishnan, Hari

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2019
Publication Date: 8/29/2019
Citation: Miranda, C.A., Xu, Q., Oehrle, N.W., Islam, N., Garrett, W.M., Natarajan, S.S., Gillman, J.D., Krishnan, H.B. 2019. Proteomic comparison of three extraction methods reveals the abundance of protease inhibitors in the seeds of grass pea, a unique orphan legume. Journal of Agricultural and Food Chemistry. 67(37):10296-10305. https://doi.org/10.1021/acs.jafc.9b04307.
DOI: https://doi.org/10.1021/acs.jafc.9b04307

Interpretive Summary: Grass pea is a drought tolerant legume that is grown for human consumption or forage in many arid and semiarid regions. Like soybean, grass pea seeds are rich in protein and can serve as a highly nutritious, inexpensive, and well-balanced source of human dietary protein. However, both soybean and grass pea contain prominent protease inhibitors that are anti-nutritional factors. These protease inhibitors affect protein digestibility and hence lowering or eliminating anti-nutritional factors has been an important target for plant breeders. In this study, we have conducted the first proteomic study of grass pea seeds. Additionally, we have evaluated three separate protein extraction procedures to isolate anti-nutritional factors from grass pea seeds. Results from our study will assist plant breeders and biotechnologists to develop legumes that are devoid of anti-nutritional factors so that we can improve the overall quality of legume seed proteins.

Technical Abstract: Grass pea is an orphan legume that is grown in many places in the world. It is a high protein, drought tolerant legume that is capable of surviving extreme environmental challenges and can be a sole food source during famine. However, grass pea produces the neurotoxin ß-N-Oxalyl-l-a,ß-diaminopropionic acid (ß-ODAP) which can cause a neurological disease. This crop is promising as a food source for both animals and humans if ß-ODAP levels and other anti-nutritional factors such as protease inhibitors are lowered or removed. To understand more about these proteins, a proteomic analysis of grass pea was conducted using three different extraction methods to determine which was more efficient at isolating anti-nutritional factors. Seed proteins extracted with Tris-buffered saline (TBS), 30% ethanol, and 50% isopropanol were identified by mass spectrometry, resulting in the documentation of the most abundant proteins for each extraction method. Mass spectrometry spectral data and BLAST2GO analysis led to the identification of 1376 proteins from all extraction methods. The molecular function of the extracted proteins revealed distinctly different protein functional profiles. The majority of the TBS-extracted proteins were annotated with nutrient reservoir activity, while the isopropanol extraction yielded the highest percentage of endopeptidase proteinase inhibitors. Our results demonstrate that the 50% isopropanol extraction method was the most efficient at isolating antinutritional factors including protease inhibitors.