Identifying Protease Fingerprints from Biological Samples

Proteases are a critically important group of proteins with a role in the activation and control of many biological processes, including those that underlie many human diseases. The human genome gives over approximately 3% of its coding space to the genes that code for protease proteins, 641 genes in all. This represents a significant proportion of the total gene complement, which gives some indication as to the importance of these regulator proteins within the body⁽¹⁾.

The key role of proteases in modulating metabolic pathways

The proteolytic cleavage of proteins mediated by proteases is a highly finessed control mechanism capable of delicate and precise control over the rate of a reaction, which is likely one of the key reasons that it has been adopted by evolution as the control mechanism of choice. The mode by which a protease activates or inactivates a target protein can take many forms, including cleavage of a latent protein to expose its active site, thus turning it on, or the cleavage of an active protein rendering it vulnerable to protein degradation, thus downregulating or turning it off. This delicate level of control mediated by proteases is central to the ability of organisms from bacteria to plants to people in the regulation of their biological and metabolic pathways, making proteases the linchpin of many biological processes and of great interest to scientists studying everything from human disease, through to the manufacture of high-value proteins in culture.

High throughput screening of native protease activity has been a challenge

While our knowledge of proteases has been expanded dramatically thanks to advances in proteomics, the technology for high-throughput multiplexed screening of protease activity within their native environment has lagged, slowing progress in our understanding of the impact of proteases, and protease activity across multiple fields of study.

A recent (2021) paper in Nature Communications by Uliana et al⁽²⁾ has sought to address this deficiency, describing for the first time a simple method for the profiling of protease activity. The author’s method is fast, cheap, easy to multiplex, and produces an accurate fingerprint of protease activity from biological samples. It is based on the isolation, separation, and identification of protease products from native lysates utilizing a modified 96-well FASP (Filter-Aided Sample Preparation) protocol and mass spectrometry.

FASP aids simplified protease fingerprinting for rapid insight into protease activity

The authors describe the development of a new high throughput protease screen (HTPS) based on the isolation of protease-specific peptides from native lysates. The team adapted a FASP protocol to a 96-well plate format using AcroPrep^TM  Advance filter plates with a 10 kDa molecular weight cut off. The use of 96-well AcroPrep filter plates to streamline the front-end sample preparation enables very high throughput and the profiling of up to 32 proteases in triplicate on the same plate. Following the modified FASP method the samples were analyzed using data-dependent acquisition (DDA) mass spectrometry followed by data analysis in the software suite R Studio (version 3.4.3).

Using this new protocol, the team was able to characterize 15 different proteases under physiologically relevant conditions and were able to obtain protease substrate profiles of sufficient detail to map specificity, determine cleavage entropy, and assess the effects of allosteric changes on substrate specificity, as well as enabling the design of protease probes. In total the team identified more than 160,000 unique substrate cleavages, substantially expanding the currently available protease knowledge base with just this one study.

The ability to undertake this type of high volume, high throughput study has great implications for our understanding of this important class of proteins. Providing a means to dramatically increase our understanding of the role of proteases across the multitude of biological processes in which they play a key, governing role. You can learn more about the AcroPrep Advance Filter Plates used in this protocol as well as other Pall products used by researchers across the globe on our life science research pages.