Detection and identification of antibacterial proteins in snake venoms using at-line nanofractionation coupled to LC-MS

Marija Mladic; Julien Slagboom; Jeroen Kool; Freek J Vonk; Gilles P van Wezel; Michael K Richardson

doi:10.1016/j.toxicon.2018.08.009

Detection and identification of antibacterial proteins in snake venoms using at-line nanofractionation coupled to LC-MS

Toxicon. 2018 Dec 1:155:66-74. doi: 10.1016/j.toxicon.2018.08.009. Epub 2018 Aug 31.

Authors

Marija Mladic¹, Julien Slagboom², Jeroen Kool³, Freek J Vonk⁴, Gilles P van Wezel⁵, Michael K Richardson⁶

Affiliations

¹ Animal Sciences and Health, Institute of Biology Leiden, University of Leiden, Sylviusweg 72, 2333BE, Leiden, The Netherlands. Electronic address: m.mladic@biology.leidenuniv.nl.
² Animal Sciences and Health, Institute of Biology Leiden, University of Leiden, Sylviusweg 72, 2333BE, Leiden, The Netherlands. Electronic address: julien_slagboom@hotmail.com.
³ Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands. Electronic address: j.kool@vu.nl.
⁴ NCB Naturalis, Sylviusweg 72, 2333BE, Leiden, The Netherlands. Electronic address: research.vonk@naturalis.nl.
⁵ Molecular Biotechnology and Health, Institute of Biology Leiden, University of Leiden, Sylviusweg 72, 2333BE, Leiden, The Netherlands. Electronic address: g.wezel@biology.leidenuniv.nl.
⁶ Animal Sciences and Health, Institute of Biology Leiden, University of Leiden, Sylviusweg 72, 2333BE, Leiden, The Netherlands. Electronic address: m.k.richardson@biology.leidenuniv.nl.

PMID: 30172790
DOI: 10.1016/j.toxicon.2018.08.009

Abstract

This study describes the application of at-line nanofractionation to the screening of snake venoms for antibacterial activity against Gram-positive and Gram-negative bacteria, the detection of proteins of interest, and their partial or full identification. A method was developed to identify bioactive peptides in crude snake venoms based on reversed-phase liquid chromatography (LC), with parallel nanofractionation onto 384-well plates and mass spectrometry (MS). Bioactivity assays were based on a resazurin-reduction assay. Accurate masses of the bioactive peptides were determined, and peptides were then identified via nanoLC-MS/MS analysis of tryptic digests, allowing full or partial identification of the bioactive proteins. Crude venoms from 41 species were screened for their antibacterial bioactivity. Venoms showing the highest activity were further screened using at-line nanofractionation, which resulted in the elucidation of 28 bioactive proteins.

Keywords: Antibacterial activity; Liquid chromatography-mass spectrometry; Resazurin-reduction assay; Snake venom.

MeSH terms

Animals
Anti-Bacterial Agents / analysis
Anti-Bacterial Agents / chemistry*
Chemical Fractionation / methods
Chromatography, Reverse-Phase / methods*
Mass Spectrometry / methods*
Nanotechnology / methods*
Peptides / analysis
Peptides / chemistry
Proteins / analysis
Proteins / chemistry*
Snake Venoms / chemistry*
Snakes

Substances

Anti-Bacterial Agents
Peptides
Proteins
Snake Venoms