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

Abstract

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. Graphical abstractᅟ Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1253-x) contains supplementary material, which is available to authorized users.

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

“…The methodology applied here facilitated the rapid identification and fractionation of coagulopathic toxins, which resulted in the identification of anticoagulant venom activities previously being masked by net procoagulant effects of the crude venom, and also detected a number of anticoagulant PLA 2 s not previously known to exhibit anticoagulant activities. Success here, alongside the recent development of other small-scale mediumthroughput bioassays focused on characterizing other relevant toxin activities [21,[84][85][86][87] means that such assays could be used interchangeably for understanding venom function and pathology-ultimately providing a 'bioassay toolkit' for deconvoluting venom mixtures. These approaches will undoubtedly facilitate new research focused on improving snakebite therapy.…”

Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins

“…Nanofractionation analytics, which is a recently developed high resolution and high throughput format of traditional bioassay-guided fractionation, is regarded as an effective method for screening complex bioactive mixtures such as venoms to rapidly identify and in parallel directly characterize separated venom toxins biochemically (i.e., for selected bioactivities), by combing reversed-phase liquid chromatography (RPLC) with parallel post-column bioassays, mass spectrometry (MS) and proteomics analysis [50][51][52]. In this paper, the coagulopathic properties of various snakes from the medically important viper subfamily Viperinae (Echis carinatus, E. ocellatus, Daboia russelii and Bitis arietans) were evaluated using nanofractionation analytics in combination with a high-throughput coagulation assay, and the inhibitory capabilities of varespladib, marimastat, dimercaprol and DMPS against the coagulopathic toxicities of the resulting snake venom fractions revealed.…”

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation

“…By applying a more shallow (60 min) gradient, the leupeptin diastereoisomers were separated with baseline resolution, allowing fractionation and microarray bioactivity testing of the individual diastereoisomers (Figure 4). When comparing picofractionation bioassay chromatograms with those given in the nanofractionation study of Zietek et al, 38 the picofractionation results do not give a better resolution. Improvement in separation resolution of venom proteins, however, was not the goal of the current study.…”

“…In order to evaluate whether the microarray bioassay can accurately determine inhibitory properties of bioactives, IC50 values of two potent plasmin inhibitors, leupeptin and aprotinin, were determined and compared with IC50 values obtained using the standard 384-well plate reader bioassay. 38 Determination of IC50 values was performed with the microarray bioassay on dried inhibitor spots that were spotted on a MTMOS coated glass slide. Leupeptin and aprotinin were dispensed at ten different concentrations ranging from 0 to 400 μM and from 0 to 30 μM, respectively.…”