Qualitative Profiling of Venom Toxins in the Venoms of Several Bothrops Species Using High-Throughput Venomics and Coagulation Bioassaying.

Envenoming resulting from snakebites is recognized as a priority neglected tropical disease by The World Health Organization. The Bothrops genus, consisting of different pitviper species, is considered the most medically significant taxa in Central and South America. Further research into Bothrops venom composition is important to aid in the development of safer and more effective snakebite treatments. In addition, the discovery of Bothrops toxins that could potentially be used for medical or diagnostic purposes is of interest to the pharmaceutical industry. This study aimed to employ high-throughput (HT) venomics to qualitatively analyze venom composition while utilizing coagulation bioassays for identifying coagulopathic toxins and characterizing coagulopathic activity in various Bothrops venoms. Using the recently demonstrated HT venomics workflow in combination with post-column coagulopathic bioassaying, focus was placed at anticoagulant toxins. Well-known procoagulant toxins were also investigated, taking into account that using the HT venomics workflow, procoagulant toxins are especially prone to denaturation during the reversed-phase chromatographic separations performed in the workflow. The findings revealed that the venoms of B. atrox and B. jararaca harbored procoagulant toxins, whereas those of B. alternatus and B. neuwiedi contained both procoagulant and anticoagulant toxins. In general, anticoagulation was associated with phospholipases A2s, while procoagulation was associated with snake venom metalloproteinases and snake venom serine proteases. These results showed the identification of coagulopathic venom toxins in the Bothrops venoms analyzed using multiple analytical methods that complement each other. Additionally, each venom underwent qualitative characterization of its composition.

Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients.

Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separation dimension (2D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS. This study reports proof-of-principle results of the development of an RPLC × RPLC-HRMS method using parallel gradients (2D flow rate of 0.7 mL min-1) and its comparison to shifted gradient methods (2D of 1.4 mL min-1) for the analysis of complex digests using HRMS (QExactive-Plus MS). Shifted and parallel gradients resulted in high surface coverage (SC) and effective peak capacity (SC of 0.6226 and 0.7439 and effective peak capacity of 779 and 757 in 60 min). When applied to a cell line digest sample, parallel gradients allowed higher sensitivity (e.g., average MS intensity increased by a factor of 3), allowing for a higher number of identifications (e.g., about 2600 vs 3900 peptides). In addition, reducing the modulation time to 10 s significantly increased the number of MS/MS events that could be performed. When compared to a 1D-RPLC method, parallel RPLC × RPLC-HRMS methods offered a higher separation performance (FHWH from 0.12 to 0.018 min) with limited sensitivity losses resulting in an increase of analyte identifications (e.g., about 6000 vs 7000 peptides and 1500 vs 1990 proteins).