蛋白酶不仅在生理过程中发挥着至关重要的作用，而且在病理过程中也发挥着至关重要的作用，例如癌症、炎症、关节炎、阿尔茨海默病和感染等。它们切割肽的能力可用于广泛的生物技术目的。为了有效地做到这一点，必须找到满足必要要求的氨基酸序列，包括特异性、选择性、切割动力学或合成可及性等相互依赖的因素。来自蛋白酶天然底物的切割序列在特异性和选择性方面可能不是最佳的，这就是为什么这些序列经常需要艰苦且有时不成功的优化，例如通过单个氨基酸的迭代交换。因此，在这里，我们描述了蛋白酶敏感接头（PSL）的系统设计——由目标蛋白酶特异性切割的肽序列——以质谱为基础，从基于蛋白质组的肽库中确定目标蛋白酶特异性切割位点。它包括一个用于识别定制 PSL 序列、其优化、合成和验证的程序，并引入了一个可以指示任意氨基酸序列中数百种酶的潜在切割位点的程序。因此，我们以基质金属蛋白酶 13 (MMP13) 为例介绍了 PSL 设计。本介绍可以作为指南，有助于大大加速蛋白酶敏感连接子在各种应用中的开发和使用。

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.