Characterisation of the forest cobra (Naja melanoleuca) venom using a multifaceted mass spectrometric-based approach

C. Ruth Wang, Alix C. Harlington, Marten F. Snel, Tara L. Pukala

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated ‘omics’ and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

Original languageEnglish
Article number140992
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Issue number2
Publication statusPublished or Issued - 1 Feb 2024


  • Forest cobra
  • Mass spectrometry
  • Proteomics
  • Snake venom proteins

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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