Utilizing liquid chromatography-mass spectrometry to map targeting of snake venom components by antivenom

Preclinical efficacy testing is an essential aspect of evaluating quality of antivenoms (AVs). Recent years have witnessed a surge in development of in vitro methods to replace or reduce reliance on the standard mouse lethality assay. In this study, we propose a novel, reversed phase liquid chromatography-mass spectrometry (RPLC-MS)-based platform for monitoring AV activity on venom components under the WHO recommended in solution AV testing conditions. The method is simple and requires only 5 μg of venom and 389 μg AV for analysis. In addition to the identification and quantification of venom components that are effectively targeted by AV and those that are not, the MS coupling also enables, for the first time, the investigation of the impact of glycosylation - a post-translational modification (PTM) on AV activity. The ability of the platform to analyse intact toxins and facilitating direct comparison of peaks between the venom and the AV incubated samples allows for a simple MS1-based relative quantitation, circumventing the challenges associated with quantitation using bottom-up tandem MS studies for non-model organisms. Further, it also allows for quantitation of total antibodies in AV against individual toxins. The method also allows us to investigate time dependent AV activity, thus enabling studies of the kinetics of AV action on individual venom components. Venoms of Cobra and Russell's viper have been analyzed to demonstrate that the proposed LC-MS method offers a simple, specific, cost-effective, and quantitative approach for screening new or established antivenoms, paving the way for the next generation antivenomics.