Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms

Barbara M. Zietek, Morwarid Mayar, Julien Slagboom, Ben Bruyneel, Freek J. Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)5751–5763
Number of pages13
JournalAnalytical and Bioanalytical Chemistry
Issue number23
Early online date9 Aug 2018
Publication statusPublished - Sept 2018


Acknowledgments The authors acknowledge ARIADME, a European FP7 ITN Community’s Seventh Framework Programme under Grant Agreement No. 607517. Many thanks to Bernhard Richter and Meet Poddar for the snake photos provided via Shutterstock.

FundersFunder number
Seventh Framework Programme607517
Seventh Framework Programme


    • Bioassay
    • Mass spectrometry
    • Metalloproteinase
    • Nanofractionation
    • Plasmin
    • Snake venoms


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