High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma
Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spe...
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doaj-56f4355b40ca4683aca26438059490022020-11-25T03:02:40ZengMDPI AGMolecules1420-30492020-09-01254071407110.3390/molecules25184071High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in PlasmaElisa Maffioli0Zhenze Jiang1Simona Nonnis2Armando Negri3Valentina Romeo4Christopher B. Lietz5Vivian Hook6Giuseppe Ristagno7Giuseppe Baselli8Erik B. Kistler9Federico Aletti10Anthony J. O’Donoghue11Gabriella Tedeschi12Department of Veterinary Medicine, University of Milano, 20133 Milano, ItalySkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USADepartment of Veterinary Medicine, University of Milano, 20133 Milano, ItalyDepartment of Veterinary Medicine, University of Milano, 20133 Milano, ItalyDepartment of Veterinary Medicine, University of Milano, 20133 Milano, ItalySkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USASkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USADepartment of Pathophysiology and Transplantation, University of Milan, 20133 Milan, ItalyDipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, 20133 Milan, ItalyDepartment of Anesthesiology & Critical Care, University of California San Diego, La Jolla, CA 92093, USADepartment of Bioengineering, University of California San Diego, La Jolla, CA 92093, USASkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USADepartment of Veterinary Medicine, University of Milano, 20133 Milano, ItalyProteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.https://www.mdpi.com/1420-3049/25/18/4071peptidomicsmass spectrometryplasmaaminopeptidasecarboxypeptidaseendoprotease |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Elisa Maffioli Zhenze Jiang Simona Nonnis Armando Negri Valentina Romeo Christopher B. Lietz Vivian Hook Giuseppe Ristagno Giuseppe Baselli Erik B. Kistler Federico Aletti Anthony J. O’Donoghue Gabriella Tedeschi |
spellingShingle |
Elisa Maffioli Zhenze Jiang Simona Nonnis Armando Negri Valentina Romeo Christopher B. Lietz Vivian Hook Giuseppe Ristagno Giuseppe Baselli Erik B. Kistler Federico Aletti Anthony J. O’Donoghue Gabriella Tedeschi High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma Molecules peptidomics mass spectrometry plasma aminopeptidase carboxypeptidase endoprotease |
author_facet |
Elisa Maffioli Zhenze Jiang Simona Nonnis Armando Negri Valentina Romeo Christopher B. Lietz Vivian Hook Giuseppe Ristagno Giuseppe Baselli Erik B. Kistler Federico Aletti Anthony J. O’Donoghue Gabriella Tedeschi |
author_sort |
Elisa Maffioli |
title |
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma |
title_short |
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma |
title_full |
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma |
title_fullStr |
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma |
title_full_unstemmed |
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma |
title_sort |
high-resolution mass spectrometry-based approaches for the detection and quantification of peptidase activity in plasma |
publisher |
MDPI AG |
series |
Molecules |
issn |
1420-3049 |
publishDate |
2020-09-01 |
description |
Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock. |
topic |
peptidomics mass spectrometry plasma aminopeptidase carboxypeptidase endoprotease |
url |
https://www.mdpi.com/1420-3049/25/18/4071 |
work_keys_str_mv |
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