Deconvolving multiplexed protease signatures with substrate reduction and activity clustering

Proteases are multifunctional, promiscuous enzymes that degrade proteins as well as peptides and drive important processes in health and disease. Current technology has enabled the construction of libraries of peptide substrates that detect protease activity, which provides valuable biological information. An ideal library would be orthogonal, such that each protease only hydrolyzes one unique substrate, however this is impractical due to off-target promiscuity (i.e., one protease targets multiple different substrates). Therefore, when a library of probes is exposed to a cocktail of proteases, each protease activates multiple probes, producing a convoluted signature. Computational methods for parsing these signatures to estimate individual protease activities primarily use an extensive collection of all possible protease-substrate combinations, which require impractical amounts of training data when expanding to search for more candidate substrates. Here we provide a computational method for estimating protease activities efficiently by reducing the number of substrates and clustering proteases with similar cleavage activities into families. We envision that this method will be used to extract meaningful diagnostic information from biological samples.Author summary: The activity of enzymatic proteins, which are called proteases, drives numerous important processes in health and disease: including cancer, immunity, and infectious disease. Many labs have developed useful diagnostics by designing sensors that measure the activity of these proteases. However, if we want to detect multiple proteases at the same time, it becomes impractical to design sensors that only detect one protease. This is due to a phenomenon called protease promiscuity, which means that proteases will activate multiple different sensors. Computational methods have been created to solve this problem, but the challenge is that these often require large amounts of training data. Further, completely different proteases may be detected by the same subset of sensors. In this work, we design a computational method to overcome this problem by clustering similar proteases into "subfamilies", which increases estimation accuracy. Further, our method tests multiple combinations of sensors to maintain accuracy while minimizing the number of sensors used. Together, we envision that this work will increase the amount of useful information we can extract from biological samples, which may lead to better clinical diagnostics.