Proteomic Comparison of Three Extraction Methods Reveals the Abundance of Protease Inhibitors in the Seeds of Grass Pea, a Unique Orphan Legume

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 β-N-oxalyl-L-α,β-diaminopropionic acid
(β-ODAP), which can cause a neurological disease. This crop
is promising as a food source for both animals and humans if β-ODAP
levels and other antinutritional 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
antinutritional 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.

山黧豆（Grass pea）是一种孤儿豆科作物，在全球诸多区域均有种植。它是一种高蛋白、耐旱的豆科植物，能够耐受极端环境胁迫，在饥荒时期可作为单一食物来源。然而，山黧豆会产生神经毒素β-N-草酰-L-α,β-二氨基丙酸（β-N-oxalyl-L-α,β-diaminopropionic acid，简称β-ODAP），该毒素可引发神经系统疾病。若能降低或去除β-ODAP与蛋白酶抑制剂等其他抗营养因子，该作物有望成为人类与动物的优质食物来源。为深入解析相关蛋白，研究人员开展了山黧豆的蛋白质组学分析，采用三种不同提取方法以筛选分离抗营养因子的最优方案。经Tris缓冲盐水（TBS）、30%乙醇及50%异丙醇提取的种子蛋白，通过质谱（mass spectrometry）鉴定，明确了各提取方法下的丰度最高蛋白。结合质谱光谱数据与BLAST2GO分析，研究从所有提取方法中共鉴定出1376种蛋白质。提取蛋白的分子功能分析显示出截然不同的蛋白功能谱：经Tris缓冲盐水提取的蛋白大多被注释为具有营养储存活性，而异丙醇提取法获得的内肽酶蛋白酶抑制剂占比最高。本研究结果表明，50%异丙醇提取法在分离包括蛋白酶抑制剂在内的抗营养因子方面效率最高。