Multiplex substrate profiling by mass spectrometry (MSP-MS): A versatile peptide digestion technique for the study of proteases — ASN Events
  1. Schedule
  2. SESSION 11: Antiinfectives
  3. Multiplex substrate profiling by mass spectrometry (MSP-MS): A versatile peptide digestion technique for the study of proteases

Multiplex substrate profiling by mass spectrometry (MSP-MS): A versatile peptide digestion technique for the study of proteases (#58)

Anthony J O'Donoghue 1 , Zhenze Jiang 1 , Michael Yoon 1 , Lawrence Liu 1 , Sam Ivry 2 , Vivian Hook 1 , Matthew Bogyo 3 , Charles S Craik 2
  1. UCSD, La Jolla, CA, United States
  2. Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA
  3. Pathology , Stanford University, Stanford, CA, USA

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.

  1. O'Donoghue AJ, Eroy-Reveles AA, Knudsen GM, Ingram J, Zhou M, Statnekov JB, Greninger AL, Hostetter DR, Qu G, Maltby DA, Anderson MO, Derisi JL, McKerrow JH, Burlingame AL, Craik CS. Global identification of peptidase specificity by multiplex substrate profiling. Nat Methods. 2012 Nov;9(11):1095-100. doi: 10.1038/nmeth.2182. Epub 2012 Sep 30.
  2. Li H, O'Donoghue AJ, van der Linden WA, Xie SC, Yoo E, Foe IT, Tilley L, Craik CS, da Fonseca PC, Bogyo M. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature. 2016 Feb 11;530(7589):233-6. doi: 10.1038/nature16936.
  3. Ivry SL, Sharib JM, Dominguez DA, Roy N, Hatcher SE, Yip-Schneider MT, Schmidt CM, Brand RE, Park WG, Hebrok M, Kim GE, O'Donoghue AJ, Kirkwood KS, Craik CS. Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts. Clin Cancer Res. 2017 Aug 15;23(16):4865-4874. doi: 10.1158/1078-0432.CCR-16-2987. Epub 2017 Apr 19.
  4. Yoon MC, Solania A, Jiang Z, Christy MP, Podvin S, Mosier C, Lietz CB, Ito G, Gerwick WH, Wolan DW, Hook G, O'Donoghue AJ, Hook V. Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions. ACS Chem Biol. 2021 Sep 17;16(9):1628-1643. doi: 10.1021/acschembio.1c00138. Epub 2021 Aug 20.
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