{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T07:44:55Z","timestamp":1768290295875,"version":"3.49.0"},"reference-count":39,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2024,4,7]],"date-time":"2024-04-07T00:00:00Z","timestamp":1712448000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["[221712\/Z\/20\/Z]"],"award-info":[{"award-number":["[221712\/Z\/20\/Z]"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>This study provides a new methodology for the rapid analysis of numerous venom samples in an automated fashion. Here, we use LC-MS (Liquid Chromatography\u2013Mass Spectrometry) for venom separation and toxin analysis at the accurate mass level combined with new in-house written bioinformatic scripts to obtain high-throughput results. This analytical methodology was validated using 31 venoms from all members of a monophyletic clade of Australian elapids: brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). In a previous study, we revealed extensive venom variation within this clade, but the data was manually processed and MS peaks were integrated into a time-consuming and labour-intensive approach. By comparing the manual approach to our new automated approach, we now present a faster and more efficient pipeline for analysing venom variation. Pooled venom separations with post-column toxin fractionations were performed for subsequent high-throughput venomics to obtain toxin IDs correlating to accurate masses for all fractionated toxins. This workflow adds another dimension to the field of venom analysis by providing opportunities to rapidly perform in-depth studies on venom variation. Our pipeline opens new possibilities for studying animal venoms as evolutionary model systems and investigating venom variation to aid in the development of better antivenoms.<\/jats:p>","DOI":"10.3390\/toxins16040181","type":"journal-article","created":{"date-parts":[[2024,4,8]],"date-time":"2024-04-08T06:04:58Z","timestamp":1712556298000},"page":"181","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Studying Venom Toxin Variation Using Accurate Masses from Liquid Chromatography\u2013Mass Spectrometry Coupled with Bioinformatic Tools"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3922-8099","authenticated-orcid":false,"given":"Luis L.","family":"Alonso","sequence":"first","affiliation":[{"name":"Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA), 1012 WX Amsterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5187-7342","authenticated-orcid":false,"given":"Jory","family":"van Thiel","sequence":"additional","affiliation":[{"name":"Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Institute of Biology Leiden, Leiden University, 2333 BE Leiden, The Netherlands"},{"name":"Naturalis Biodiversity Center, 2333 CR Leiden, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2929-1086","authenticated-orcid":false,"given":"Julien","family":"Slagboom","sequence":"additional","affiliation":[{"name":"Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA), 1012 WX Amsterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nathan","family":"Dunstan","sequence":"additional","affiliation":[{"name":"Venom Supplies Pty. Ltd., Tanunda, SA 5352, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2211-3930","authenticated-orcid":false,"given":"Cassandra M.","family":"Modahl","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Timothy N. W.","family":"Jackson","sequence":"additional","affiliation":[{"name":"Australian Venom Research Unit, Department of Biochemistry and Pharmacology, University of Melbourne, Parkville, VIC 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8270-6979","authenticated-orcid":false,"given":"Saer","family":"Samanipour","sequence":"additional","affiliation":[{"name":"Van\u2018t Hof Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0011-5612","authenticated-orcid":false,"given":"Jeroen","family":"Kool","sequence":"additional","affiliation":[{"name":"Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA), 1012 WX Amsterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Malhotra, A. (2017). Evolution of Venomous Animals and Their Toxins, Springer.","DOI":"10.1007\/978-94-007-6458-3"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.tree.2012.10.020","article-title":"Complex cocktails: The evolutionary novelty of venoms","volume":"28","author":"Casewell","year":"2013","journal-title":"Trends Ecol. Evol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1038\/379537a0","article-title":"Diet and snake venom evolution","volume":"379","author":"Daltry","year":"1996","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2443","DOI":"10.1098\/rspb.2009.0048","article-title":"Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution","volume":"276","author":"Barlow","year":"2009","journal-title":"Proc. R. Soc. B Biol. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e2015579118","DOI":"10.1073\/pnas.2015579118","article-title":"Phylogenetically diverse diets favor more complex venoms in North American pitvipers","volume":"118","author":"Holding","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Ward-Smith, H., Arbuckle, K., Naude, A., and W\u00fcster, W. (2020). Fangs for the Memories? A Survey of Pain in Snakebite Patients Does Not Support a Strong Role for Defense in the Evolution of Snake Venom Composition. Toxins, 12.","DOI":"10.3390\/toxins12030201"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1126\/science.abb9303","article-title":"Convergent evolution of pain-inducing defensive venom components in spitting cobras","volume":"371","author":"Kazandjian","year":"2022","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1823","DOI":"10.1111\/brv.12865","article-title":"Convergent evolution of toxin resistance in animals","volume":"97","author":"Khan","year":"2022","journal-title":"Biol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1016\/j.tips.2020.05.006","article-title":"Causes and Consequences of Snake Venom Variation","volume":"41","author":"Casewell","year":"2020","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"van Thiel, J., Alonso, L.L., Slagboom, J., Dunstan, N., Wouters, R.M., Modahl, C.M., Vonk, F.J., Jackson, T.N., and Kool, J. (2023). Highly Evolvable: Investigating Interspecific and Intraspecific Venom Variation in Taipans (Oxyuranus spp.) and Brown Snakes (Pseudonaja spp.). Toxins, 15.","DOI":"10.3390\/toxins15010074"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Jackson, T.N.W., Koludarov, I., Ali, S.A., Dobson, J., Zdenek, C.N., Dashevsky, D., Op den Brouw, B., Masci, P.P., Nouwens, A., and Josh, P. (2016). Rapid Radiations and the Race to Redundancy: An Investigation of the Evolution of Australian Elapid Snake Venoms. Toxins, 8.","DOI":"10.3390\/toxins8110309"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Avella, I., W\u00fcster, W., Luiselli, L., and Mart\u00ednez-Freir\u00eda, F. (2022). Toxic Habits: An Analysis of General Trends and Biases in Snake Venom Research. Toxins, 14.","DOI":"10.3390\/toxins14120884"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9205","DOI":"10.1073\/pnas.1405484111","article-title":"Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms","volume":"111","author":"Casewell","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Smith, C.F., Nikolakis, Z.L., Ivey, K., Perry, B.W., Schield, D.R., Balchan, N.R., Parker, J., Hansen, K.C., Saviola, A.J., and Castoe, T.A. (2023). Snakes on a plain: Biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake. BMC Biol., 21.","DOI":"10.1186\/s12915-023-01626-x"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"17063","DOI":"10.1038\/nrdp.2017.63","article-title":"Snakebite envenoming","volume":"3","author":"Calvete","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1768","DOI":"10.1016\/j.jprot.2011.06.019","article-title":"Research strategies to improve snakebite treatment: Challenges and progress","volume":"74","author":"Harrison","year":"2011","journal-title":"J. Proteom."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"103443","DOI":"10.1016\/j.jprot.2019.103443","article-title":"Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka","volume":"207","author":"Pla","year":"2019","journal-title":"J. Proteom."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/s41570-022-00393-7","article-title":"The chemistry of snake venom and its medicinal potential","volume":"6","author":"Oliveira","year":"2022","journal-title":"Nat. Rev. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1586\/epr.11.61","article-title":"Proteomic tools against the neglected pathology of snake bite envenoming","volume":"8","author":"Calvete","year":"2011","journal-title":"Expert. Rev. Proteom."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2088","DOI":"10.1093\/gbe\/evu166","article-title":"Restriction and Recruitment\u2014Gene Duplication and the Origin and Evolution of Snake Venom Toxins","volume":"6","author":"Hargreaves","year":"2014","journal-title":"Genome Biol. Evol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"101066","DOI":"10.1016\/j.mam.2022.101066","article-title":"Identification and characterization of post-translational modifications: Clinical implications","volume":"86","author":"Hermann","year":"2022","journal-title":"Mol. Asp. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1734","DOI":"10.1021\/acs.jproteome.2c00780","article-title":"High-Throughput Venomics","volume":"22","author":"Slagboom","year":"2023","journal-title":"J. Proteome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/0041-0101(79)90245-9","article-title":"The lethality in mice of dangerous Australian and other snake venom","volume":"17","author":"Broad","year":"1979","journal-title":"Toxicon"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"119","DOI":"10.5694\/mja17.00094","article-title":"The Australian Snakebite Project, 2005\u20132015 (ASP-20)","volume":"207","author":"Johnston","year":"2017","journal-title":"Med. J. Aust."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1016\/j.toxicon.2006.11.026","article-title":"Antivenom use, premedication and early adverse reactions in the management of snake bites in rural Papua New Guinea","volume":"49","author":"Williams","year":"2007","journal-title":"Toxicon"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/s40409-017-0135-6","article-title":"It is time for top-down venomics","volume":"23","author":"Melani","year":"2017","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3884","DOI":"10.1021\/acs.analchem.6b04857","article-title":"Width Based Quantitation of Chromatographic Peaks: Principles and Principal Characteristics","volume":"89","author":"Kadjo","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2128","DOI":"10.1016\/j.jprot.2012.01.006","article-title":"Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: Role of neurotoxic and procoagulant effects in venom toxicity","volume":"75","author":"Herrera","year":"2012","journal-title":"J. Proteom."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Tasoulis, T., Silva, A., Veerati, P.C., Baker, M., Hodgson, W.C., Dunstan, N., and Isbister, G.K. (2020). Intra-Specific Venom Variation in the Australian Coastal Taipan Oxyuranus scutellatus. Toxins, 12.","DOI":"10.3390\/toxins12080485"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Skejic, J., Steer, D.L., Dunstan, N., and Hodgson, W.C. (2018). Venoms of related mammal-eating species of taipans (Oxyuranus) and brown snakes (Pseudonaja) differ in composition of toxins involved in mammal poisoning. Evol. Biol.","DOI":"10.1101\/378141"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1111\/j.1432-1033.1976.tb10833.x","article-title":"Taipoxin, an Extremely Potent Presynaptic Neurotoxin from the Venom of the Australian Snake Taipan (Oxyuranus s. scutellatus)","volume":"68","author":"Fohlman","year":"1976","journal-title":"Eur. J. Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1074\/mcp.M600419-MCP200","article-title":"The Diversity of Bioactive Proteins in Australian Snake Venoms","volume":"6","author":"Birrell","year":"2007","journal-title":"Mol. Cell. Proteom."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.jprot.2015.11.019","article-title":"Deep venomics of the Pseudonaja genus reveals inter-and intra-specific variation","volume":"133","author":"Reeks","year":"2016","journal-title":"J. Proteom."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.jprot.2016.05.027","article-title":"Proteomic comparisons of venoms of long-term captive and recently wild-caught Eastern brown snakes (Pseudonaja textilis) indicate venom does not change due to captivity","volume":"144","author":"McCleary","year":"2016","journal-title":"J. Proteom."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Modahl, C.M., Han, S.X., Van Thiel, J., Vaz, C., Dunstan, N.L., Frietze, S., Jackson, T.N., Mackessy, S.P., and Kini, R.M. (2023). Distinct regulatory networks control toxin gene expression in elapid and viperid snakes. BMC Genom., 25.","DOI":"10.1101\/2023.02.13.528252"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2658","DOI":"10.1021\/acs.jproteome.6b00217","article-title":"Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes","volume":"15","author":"Zelanis","year":"2016","journal-title":"J. Proteome Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6554","DOI":"10.1002\/pmic.200600263","article-title":"Post-translational modification accounts for the presence of varied forms of nerve growth factor in Australian elapid snake venoms","volume":"6","author":"Earl","year":"2006","journal-title":"Proteomics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.cca.2004.04.004","article-title":"The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases","volume":"138","author":"Ramos","year":"2004","journal-title":"Comp. Biochem. Physiol. Part C Toxicol. Pharmacol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2621","DOI":"10.3390\/toxins5122621","article-title":"Venom Down Under: Dynamic Evolution of Australian Elapid Snake Toxins","volume":"5","author":"Jackson","year":"2013","journal-title":"Toxins"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/4\/181\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:24:29Z","timestamp":1760106269000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/4\/181"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,7]]},"references-count":39,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["toxins16040181"],"URL":"https:\/\/doi.org\/10.3390\/toxins16040181","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,7]]}}}