Large-scale mass spectrometry-based peptidomics for bioactive-peptide discovery is relatively unexplored because of challenges in intracellular peptide extraction and small-peptide identification. Here, we present an analytical pipeline for large-scale intracellular peptidomics of Lactococcus lactis. It entails an optimized sample preparation protocol for L. lactis, used as an “enzyme complex” to digest β-casein, an extraction method for its intracellular peptidome, and a peptidomics data analysis and visualization procedure. In addition, we proofread the publicly available bioactive-peptide databases and obtained an optimized database of bioactive peptides derivable from bovine β-casein. We used the pipeline to examine cultures of L. lactis MG1363 and a set of 6 isogenic multiple peptidase mutants incubated with β-casein. We observed a clearly strain-dependent accumulation of peptides with several bioactivities, such as angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl peptidase 4 (DPP-IV)-inhibitory, and immunoregulatory functions. The results suggest that both the number of different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of L. lactis. This comprehensive pipeline offers a model for discovery of bioactive peptides in combination with other proteins and might be applicable to other bacteria.

中文翻译：

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乳酸乳球菌蛋白水解系统的编辑增加了其生物活性潜力。

由于细胞内肽提取和小肽鉴定方面的挑战，用于生物活性肽发现的大规模基于质谱的肽组学相对尚未被探索。在这里，我们提出了用于乳酸乳球菌大规模细胞内肽组学的分析管道。它需要针对乳酸乳球菌进行优化的样品制备方案，用作消化β-酪蛋白的“酶复合物”，其细胞内肽组的提取方法，以及肽组学数据分析和可视化程序。此外，我们对公开的生物活性肽数据库进行了校对，获得了牛β-酪蛋白生物活性肽的优化数据库。我们使用该流程来检查乳酸乳球菌MG1363 的培养物以及与 β-酪蛋白一起孵育的一组 6 个同基因多重肽酶突变体。我们观察到具有多种生物活性的肽明显依赖于菌株的积累，例如血管紧张素转换酶 (ACE) 抑制、二肽基肽酶 4 (DPP-IV) 抑制和免疫调节功能。结果表明，通过编辑乳酸乳球菌的蛋白水解系统，可以增加不同生物活性肽的数量和生物活性多样性。这个综合管道为发现与其他蛋白质结合的生物活性肽提供了一个模型，并且可能适用于其他细菌。