Wang H B, Zeng F, Wang Y Y, Li X, Li Y M, Wang Y F, Liu Y H, Lu F P
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, National Engineering Laboratory for Industrial Enzymes, The College of Biotechnology, Tianjin University of Science and Technology Tianjin 300457 P. R. China
RSC Adv. 2020 Oct 7;10(61):37182-37186. doi: 10.1039/d0ra04226a.
Proteases are widely used in the food industry to hydrolyze proteins and prepare bioactive peptides. Peptide mapping identification supports the application of proteases in the food industry. The site-specified peptide identification method, which was developed for site-specific proteases like trypsin, is relatively mature and reliable but cannot be applied using most industrial proteases with weak site specificity. To address this issue, the performance and reliability of the site-unspecified peptide identification method should be investigated and evaluated. In this study, tryptic hydrolysates of a single protein and a protein mixture were used to evaluate the site-unspecified identification method. The species origin of the hydrolyzed proteins was not specified in a database search, meaning that millions of protein sequences were included for calculating and matching. At least 98% of the tryptic peptides were successfully identified the site-unspecified method, demonstrating that the site-unspecified method shows promising reliability. Moreover, the site-unspecified method identified more peptides than the site-specified method, including those from the low-frequency site-unspecific hydrolysis of trypsin, suggesting that the method has strong capabilities for peptide mapping. The results indicate the applicability of the site-unspecified peptide identification method in the study of site-unspecific industrial proteases.
蛋白酶在食品工业中被广泛用于水解蛋白质并制备生物活性肽。肽图谱鉴定支持蛋白酶在食品工业中的应用。针对胰蛋白酶等位点特异性蛋白酶开发的位点特异性肽鉴定方法相对成熟且可靠，但大多数位点特异性较弱的工业蛋白酶无法应用该方法。为解决这一问题，应研究和评估非位点特异性肽鉴定方法的性能和可靠性。在本研究中，使用单一蛋白质和蛋白质混合物的胰蛋白酶水解产物来评估非位点特异性鉴定方法。在数据库搜索中未指定水解蛋白质的物种来源，这意味着计算和匹配时包含了数百万个蛋白质序列。至少98%的胰蛋白酶肽通过非位点特异性方法成功鉴定，表明非位点特异性方法具有可靠的前景。此外，非位点特异性方法比位点特异性方法鉴定出更多的肽，包括来自胰蛋白酶低频非位点特异性水解的肽，这表明该方法具有很强的肽图谱分析能力。结果表明非位点特异性肽鉴定方法在非位点特异性工业蛋白酶研究中的适用性。
