Analytical Size Exclusion Chromatography Coupled with Mass Spectrometry in Parallel with High-Throughput Venomics and Bioassaying for Venom Profiling.

Modern analytical size exclusion chromatography (SEC) is a suitable technique to separate venom toxin families according to their size characteristics. In this study, a method was developed to separate intact venom toxins from Bungarus multicinctus and Daboia russelii venoms via analytical SEC using volatile, non-salt-containing eluents for post-column mass spectrometry, coagulation bioassaying and high-throughput venomics. Two venoms were used to demonstrate the method developed. While the venom of Bungaurs multicinctus is known to exert anticoagulant effects on plasma, in this study, we showed the existence of both procoagulant toxins and anticoagulant toxins. For Daboia russelii venom, the method revealed characteristic procoagulant effects, with a 90 kDa mass toxin detected and matched with the Factor X-activating procoagulant heterotrimeric glycoprotein named RVV-X. The strong procoagulant effects for this toxin show that it was most likely eluted from size exclusion chromatography non-denatured. In conclusion, the separation of snake venom by size gave the opportunity to separate some specific toxin families from each other non-denatured, test these for functional bioactivities, detect the eluting mass on-line via mass spectrometry and identify the eluted toxins using high-throughput venomics.

Development of a high-throughput in vitro screening method for the assessment of cell-damaging activities of snake venoms.

Snakebite envenoming is a globally important public health issue that has devastating consequences on human health and well-being, with annual mortality rates between 81,000 and 138,000. Snake venoms may cause different pathological effects by altering normal physiological processes such as nervous transfer and blood coagulation. In addition, snake venoms can cause severe (local) tissue damage that may result in life-long morbidities, with current estimates pointing towards an additional 450,000 individuals that suffer from permanent disabilities such as amputations, contractions and blindness. Despite such high morbidity rates, research to date has been mainly focusing on neurotoxic and haemotoxic effects of snake venoms and considerably less on venom-induced tissue damage. The molecular mechanisms underlaying this pathology include membrane disruption and extracellular matrix degradation. This research describes methods used to study the (molecular) mechanisms underlaying venom-induced cell- and tissue damage. A selection of cellular bioassays and fluorescent microscopy were used to study cell-damaging activities of snake venoms in multi-well plates, using both crude and fractionated venoms. A panel of 10 representative medically relevant snake species was used, which cover a large part of the geographical regions most heavily affected by snakebite. The study comprises both morphological data as well as quantitative data on cell metabolism and viability, which were measured over time. Based on this data, a distinction could be made in the ways by which viper and elapid venoms exert their effects on cells. We further made an effort to characterise the bioactive compounds causing these effects, using a combination of liquid chromatography methods followed by bioassaying and protein identification using proteomics. The outcomes of this study might prove valuable for better understanding venom-induced cell- and tissue-damaging pathologies and could be used in the process of developing and improving snakebite treatments.

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins.

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM-polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms.

Supercritical fluid chromatography - Mass spectrometry in metabolomics: Past, present, and future perspectives.

Metabolomics, which consists of the comprehensive analysis of metabolites within a biological system, has been playing a growing role in the implementation of personalized medicine in modern healthcare. A wide range of analytical approaches are used in metabolomics, notably mass spectrometry (MS) combined to liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE). However, none of these methods enable a comprehensive analysis of the metabolome, due to its extreme complexity and the large differences in physico-chemical properties between metabolite classes. In this context, supercritical fluid chromatography (SFC) represents a promising alternative approach to improve the metabolome coverage, while further increasing the analysis throughput. SFC, which uses supercritical CO2 as mobile phase, leads to numerous advantages such as improved kinetic performance and lower environmental impact. This chromatographic technique has gained a significant interest since the introduction of advanced instrumentation, together with the introduction of dedicated interfaces for hyphenating SFC to MS. Moreover, new developments in SFC column chemistry (including sub-2 µm particles), as well as the use of large amounts of organic modifiers and additives in the CO2-based mobile phase, significantly extended the application range of SFC, enabling the simultaneous analysis of a large diversity of metabolites. Over the last years, several applications have been reported in metabolomics using SFC-MS - from lipophilic compounds, such as steroids and other lipids, to highly polar compounds, such as carbohydrates, amino acids, or nucleosides. With all these advantages, SFC-MS is promised to a bright future in the field of metabolomics.

Supercritical fluid chromatography as a rapid single-step method for the determination of mineral oil saturated and aromatic hydrocarbons in purified mineral oils for food and cosmetics applications.

Mineral oil hydrocarbons are used in the consumer goods sector for the elaboration of a wide range of foods and cosmetics. Traditional methods for determining their levels and composition are time consuming and laborious, besides requiring complex instrumentation. Here a simple and fast method was developed that uses columns packed with silver-modified silica in supercritical fluid chromatography with flame ionization and UV detection (SFC-FID/UV) for the determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in purified mineral oil samples. The method requires no sample preparation apart from dilution. Direct quantification of MOSH and MOAH was possible for samples with MOSH/MOAH ratios around one. For other samples deconvolution of the MOSH and MOAH humps in the FID chromatogram using the UV signal was needed since baseline separation of the two fractions could not be obtained. Validation of the method performance showed an excellent linearity (R2 > 0.9995) in the range of concentrations tested (2.5-100 mgmL-1) and a better repeatability than the standard methods (<5%). MOAH detection limits were better than 0.36% MOAH, which makes the method sufficiently sensitive for analysis of all but the highest purity mineral oils. The proposed SFC-FID/UV method was suitable for the analysis of mineral oils with viscosities and molecular weights below approximately 56 mm2s-1 and 450 gmol-1. The quantitative results of the new method were not statistically significantly different from those obtained with the standard SPE-GC-FID method where the new method has the advantages of a better repeatability, simpler operation and a significantly shortened analysis time. This new method could potentially also be used for the analysis of mineral oil contaminations in consumer products such as foods. However, in this case additional sample clean-up and preconcentration steps are needed for reducing matrix interferences from e.g. triglycerides and olefins and for improving the detection limits.

Comprehensive off-line silver phase liquid chromatography × gas chromatography with flame ionization and vacuum ultraviolet detection for the detailed characterization of mineral oil aromatic hydrocarbons.

Highly purified mineral oils used for the elaboration of pharmaceutical, food and cosmetic products can contain residual mineral oil aromatic hydrocarbons (MOAH). Quantification of the MOAH level as well as detailed characterization of the aromatic species present is important for safety evaluations and for optimization of the purification process. Two comprehensive off-line silver phase liquid chromatography × gas chromatography (AgLC × GC) methods, one with flame ionization detection (FID) and another with vacuum ultraviolet detection (VUV), were developed for MOAH analysis. The methods showed a better resolution between the MOSH and MOAH groups compared to the traditional online LC-GC methods due to the different retention mechanisms employed in the two dimensions, albeit that the gain was less than seen e.g. in edible oil analysis. An important advantage of the new comprehensive AgLC × GC methods is that the use of markers to determine the MOSH/MOAH cut-point is no longer needed, because all the eluent coming from the LC separation is transferred as narrow fractions to the GC. Due to the use of silver based stationary phases in the first separation dimension, a group-type separation of the mineral oil according to the degree of aromaticity (aliphatics, mono-aromatics and poly-aromatics) was obtained. Moreover, thanks to the use of VUV detection, the new method also delivered additional structural information on the different groups of compounds present.