An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example

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.

蛋白酶（protease）不仅在生理过程中发挥核心作用，在癌症、炎症、关节炎、阿尔茨海默病、感染等多种病理过程中同样具有关键意义。其切割肽段的能力可被应用于诸多生物技术场景。为高效达成这一目标，需筛选出符合特定要求的氨基酸序列，同时需兼顾特异性、选择性、切割动力学以及合成可行性等多项相互关联的影响因素。源自蛋白酶天然底物的切割序列，在特异性与选择性层面往往并非最优，因此通常需要通过迭代替换单个氨基酸等方式开展繁琐且有时甚至无法成功的优化工作。有鉴于此，本研究阐述了以基于蛋白质组肽库的靶向蛋白酶特异性切割位点的质谱测定结果为指导，系统设计蛋白酶敏感接头（protease sensitive linkers, PSLs）的方法——此类肽序列可被目标蛋白酶特异性切割。该方案涵盖定制化PSL序列的鉴定、优化、合成与验证流程，并推出一款可针对任意氨基酸序列预测数百种酶潜在切割位点的工具。本研究以基质金属蛋白酶13（matrix metalloproteinase 13, MMP13）为例，对PSL的设计流程进行了详细阐释。本指南可为各类应用场景中蛋白酶敏感接头的开发与应用提供参考，大幅推动相关研究与应用进程。