Nanofractionation Analytics for Comparing MALDI-MS and ESI-MS Data of Viperidae Snake Venom Toxins

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (<i>Bothrops asper</i>, <i>Crotalus atrox</i>, and <i>Daboia russelii</i>) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.