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*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)66-74
Number of pages9
JournalToxicon
Volume155
Early online date31 Aug 2018
DOIs
Publication statusPublished - 1 Dec 2018

Keywords

  • Antibacterial activity
  • Liquid chromatography-mass spectrometry
  • Resazurin-reduction assay
  • Snake venom

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