High throughput venomics
Slagboom, Julien
j.slagboom@vu.nl
Amsterdam Institute of Molecular and Life Sciences (AIMMS)
Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam
Amsterdam, The Netherlands
Derks, Rico
Center for Proteomics and Metabolomics
Leiden Universitair Medisch Centrum
Leiden, The Netherlands
Somsen, Govert W.
Amsterdam Institute of Molecular and Life Sciences (AIMMS)
Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam
Amsterdam, The Netherlands
Vonk, Freek J. Amsterdam Institute of Molecular and Life Sciences (AIMMS)
Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam
Amsterdam, The Netherlands
Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands
Casewell, Nicholas R.
Centre for Snakebite Research and Interventions
Liverpool School of Tropical Medicine
Liverpool, United Kingdom
Kool, Jeroen
Amsterdam Institute of Molecular and Life Sciences (AIMMS)
Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam
Amsterdam, The Netherlands
In this study we show high-throughput (HT) venomics capable of performing a full proteomic analysis of a snake venom within 2 days. The workflow starts with subjecting a snake venom to nanofractionation analytics, which involves liquid chromatographic separation of the toxins in a venom followed by mass spectrometry analysis and in parallel high-resolution fractionation on a 384 well-plate. After vacuum-centrifugation of the well plates to evaporate the eluents, automated tryptic digestion of all fractionated toxins is performed. Then, all digests are analysed using a fast-analytical gradient with a total runtime of 14.4 min per well, resulting in 100 nanoLC-MS/MS measurements per day. The data obtained from all wells is then automatically processed and subjected to Mascot database searching. From there using an in-house written script, all Mascot results are automatically compiled into a single Excel sheet containing all the proteomics data of an analysed snake venom. Then, another script plots each of the identified toxins in so called Protein Score Chromatograms. For this, for each toxin identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. This same script integrates the peaks in these chromatograms for semi-quantitation purposes. This new HT venomics strategy was performed on the venoms of Calloselasma rhodostoma, Echis ocellatus and Naja pallida, Bothrops asper, B. multicinctus, Crotalus atrox, Daboia russelii, N. naja, N. nigricollis, N. mossambica, and Ophiophagus hannah. Our data suggest that high throughput venomics will be a highly valuable tool for increasing the throughput by which we can define venom variation and should greatly aid the future development of new snakebite treatments