Identification of zein proteome by multi-enzyme digestion

Zeins are a class of prolamine proteins extracted from maize, and are extensively used in the food and pharmaceutical industries. Therefore, characterization of its components is essential for quality control and safety evaluation. However, zein proteins have low abundance of the trypsin targets lysine and arginine, thereby requiring enzymes of different cleavage sites for shotgun proteomic analyses. To this end, we performed in silico digestion of zein proteins using tandem combinations of trypsin/Lys-C, trypsin/chymotrypsin and trypsin/thermolysin, in order to improve protein sequence coverage and subsequent identification by LC-MS/MS analysis. Trypsin/chymotrypsin yielded the highest protein sequence coverage of up to 79.5% by the filter-aided sample preparation (FASP) method, and increased the number of detectable proteins from 11 to 35 compared to trypsin/Lys-C. Furthermore, SDS-PAGE revealed 37 proteins in the zein extract, as well as the possibility

玉米醇溶蛋白（zein）是一类从玉米中提取的醇溶谷蛋白，在食品与制药工业中应用广泛。因此，对其组分进行表征对于质量控制与安全性评价均至关重要。然而，玉米醇溶蛋白中作为胰蛋白酶酶切靶点的赖氨酸与精氨酸丰度较低，使得鸟枪蛋白质组学分析需采用具备不同酶切位点的酶类。为此，我们采用胰蛋白酶/赖氨酸内切酶（Lys-C）、胰蛋白酶/胰凝乳蛋白酶以及胰蛋白酶/嗜热菌蛋白酶的组合酶切方案，对玉米醇溶蛋白开展计算机模拟酶切，以提升蛋白质序列覆盖率，并通过液相色谱-串联质谱（LC-MS/MS）分析实现更高效的蛋白质鉴定。其中，采用辅助过滤样品制备（FASP）法结合胰蛋白酶/胰凝乳蛋白酶组合，可实现最高达79.5%的蛋白质序列覆盖率；与胰蛋白酶/赖氨酸内切酶组合相比，该方案可将可检测到的蛋白质数量从11种提升至35种。此外，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示该玉米醇溶蛋白提取物中含有37种蛋白质，同时还存在相关可能性。