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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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Autores principales: Maffioli, Elisa, Jiang, Zhenze, Nonnis, Simona, Negri, Armando, Romeo, Valentina, Lietz, Christopher B., Hook, Vivian, Ristagno, Giuseppe, Baselli, Giuseppe, Kistler, Erik B., Aletti, Federico, O’Donoghue, Anthony J., Tedeschi, Gabriella
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571063/
https://www.ncbi.nlm.nih.gov/pubmed/32899982
http://dx.doi.org/10.3390/molecules25184071
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author Maffioli, Elisa
Jiang, Zhenze
Nonnis, Simona
Negri, Armando
Romeo, Valentina
Lietz, Christopher B.
Hook, Vivian
Ristagno, Giuseppe
Baselli, Giuseppe
Kistler, Erik B.
Aletti, Federico
O’Donoghue, Anthony J.
Tedeschi, Gabriella
author_facet Maffioli, Elisa
Jiang, Zhenze
Nonnis, Simona
Negri, Armando
Romeo, Valentina
Lietz, Christopher B.
Hook, Vivian
Ristagno, Giuseppe
Baselli, Giuseppe
Kistler, Erik B.
Aletti, Federico
O’Donoghue, Anthony J.
Tedeschi, Gabriella
author_sort Maffioli, Elisa
collection PubMed
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.
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spelling pubmed-75710632020-10-28 High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma Maffioli, Elisa Jiang, Zhenze Nonnis, Simona Negri, Armando Romeo, Valentina Lietz, Christopher B. Hook, Vivian Ristagno, Giuseppe Baselli, Giuseppe Kistler, Erik B. Aletti, Federico O’Donoghue, Anthony J. Tedeschi, Gabriella Molecules Article 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. MDPI 2020-09-06 /pmc/articles/PMC7571063/ /pubmed/32899982 http://dx.doi.org/10.3390/molecules25184071 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Maffioli, Elisa
Jiang, Zhenze
Nonnis, Simona
Negri, Armando
Romeo, Valentina
Lietz, Christopher B.
Hook, Vivian
Ristagno, Giuseppe
Baselli, Giuseppe
Kistler, Erik B.
Aletti, Federico
O’Donoghue, Anthony J.
Tedeschi, Gabriella
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma
title 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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571063/
https://www.ncbi.nlm.nih.gov/pubmed/32899982
http://dx.doi.org/10.3390/molecules25184071
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