Abstract
This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC50 = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC50 = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.
Original language | English |
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Pages (from-to) | 5987-5997 |
Number of pages | 11 |
Journal | Analytical and Bioanalytical Chemistry |
Volume | 409 |
Issue number | 25 |
DOIs | |
Publication status | Published - Oct 2017 |
Funding
This research was supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organization for Scientific Research (NWO), and partly funded by the Ministry of Economic Affairs (project number 11056). The authors declare that they have no conflict of interest. This research was supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organization for Scientific Research (NWO), and partly funded by the Ministry of Economic Affairs (project number 11056).
Funders | Funder number |
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Ministerie van Economische Zaken | 11056 |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek | |
Stichting voor de Technische Wetenschappen | |
Ministry of Economic Affairs |
Keywords
- ACE inhibitors
- At-line nanofractionation
- Liquid chromatography-mass spectrometry
- Snake venoms