Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry.

Unlabelled: We report the application of a hybrid element and molecular MS configuration for the parallel absolute quantification of μHPLC-separated intact sulfur-containing venom proteins, via ICP triple quadrupole MS and S/S isotope dilution analysis, and identification by ESI-QToF-MS of the toxins of the medically important African black-necked spitting cobra, Naja nigricollis (Tanzania); New Guinea small-eyed snake, Micropechis ikaheka; and Papuan black snake, Pseudechis papuanus. The main advantage of this approach is that only one generic sulfur-containing standard is required to quantify each and all intact Cys- and/or Met-containing toxins of the venom proteome. The results of absolute quantification are in reasonably good agreement with previously reported relative quantification of the most abundant protein families. However, both datasets depart in the quantification of the minor ones, showing a tendency for this set of proteins to be underestimated in standard peptide-centric venomics approaches. The molecular identity, specific toxic activity, and concentration in the venom, are the pillars on which the toxicovenomics-aimed discovery of the most medically-relevant venom toxins, e.g. those that need to be neutralized by an effective therapeutic antivenom, should be based. The pioneering venom proteome-wide absolute quantification shown in this paper represents thus a significant advance towards this goal. The potential of ICP triple quadrupole MS in proteomics in general, and venomics in particular, is critically discussed.

Biological Significance: Animal venoms provide excellent model systems for investigating interactions between predators and prey, and the molecular mechanisms that contribute to adaptive protein evolution. On the other hand, numerous cases of snake bites occur yearly by encounters of humans and snakes in their shared natural environment. Snakebite envenoming is a serious global public health issue that affects the most impoverished and geopolitically disadvantaged rural communities in many tropical and subtropical countries. Unveiling the temporal and spatial patterns of venom variability is of fundamental importance to understand the molecular basis of envenoming, a prerequisite for developing therapeutic strategies against snakebite envenoming. Research on venoms has been continuously enhanced by advances in technology. The combined application of next-generation transcriptomic and venomic workflows has demonstrated unparalleled capabilities for venom characterization in unprecedented detail. However, mass spectrometry is not inherently quantitative, and this analytical limitation has sparked the development of methods to determine absolute abundance of proteins in biological samples. Here we show the potential of a hybrid element and molecular MS configuration for the parallel ESI-QToF-MS and ICP-QQQ detection and absolute quantification of intact sulfur-containing venom proteins via S/S isotope dilution analysis. This configuration has been applied to quantify the toxins of the medically important African snake Naja nigricollis (Tanzania), and the Papuan species Micropechis ikaheka and Pseudechis papuanus.