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.
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma / E. Maffioli, Z. Jiang, S. Nonnis, A. Negri, V. Romeo, C.B. Lietz, V. Hook, G. Ristagno, G. Baselli, E.B. Kistler, F. Aletti, A.J. O'Donoghue, G. Tedeschi. - In: MOLECULES. - ISSN 1420-3049. - 25:18(2020 Sep 06), pp. 4071.1-4071.14.
|Titolo:||High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma|
MAFFIOLI, ELISA MARGHERITA (Primo)
TEDESCHI, GABRIELLA (Corresponding)
|Parole Chiave:||aminopeptidase; carboxypeptidase; endoprotease; mass spectrometry; peptidomics; plasma|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||6-set-2020|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.3390/molecules25184071|
|Appare nelle tipologie:||01 - Articolo su periodico|