Our lab uses a mass spectrometry-based peptide digestion assay called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to study global proteolytic activity in complex biological samples. MSP-MS has enabled us to discover novel proteolytic activities in secretory vesicles, plasma, cyst fluid, and protein extracts from cancer cells, immune cells, parasites, bacteria, and fungi. We have also generated substrate specificity profiles of individual proteases from mammalian cells and numerous different microbes. This knowledge has allowed us to design fluorescent substrates, peptide inhibitors, and activity-based probes that are selective for specific proteases.
In this talk, I will discuss our recent findings on the use of MSP-MS to study proteolytic activity in premalignant pancreatic cyst fluid. We identified a novel protease that is present in premalignant cyst fluid but not in benign cyst fluid. We then developed a diagnostic reagent that can be used to quantify the levels of this protease in pancreatic cysts. This information could be used to guide surgical decision-making for patients with pancreatic cysts.
We have also used MSP-MS to design potent and selective inhibitors of human cathepsin B. Cathepsin B is a protease that is involved in neurodegenerative diseases. Our inhibitors target cathepsin B at the neutral pH conditions of the cytoplasm, where it is most pathogenic. This approach has the potential to develop new therapies for neurodegenerative diseases.
Finally, we have used MSP-MS to compare the peptide digestion patterns generated by the malaria (Plasmodium falciparum) and human proteasomes. We identified key amino acids in the substrate that are selective for the malaria enzyme. This information was used to develop a potent and selective proteasome inhibitor that eliminates parasite burden in an animal infection model without any toxicity to the host.