Unravelling Indian Cobra Venom Using 2D-LC–MS

Researchers at the Indian Institute of Technology Delhi in New Delhi, India, introduced a two-dimensional liquid chromatography–mass spectrometry (2D-LC–MS) platform for the separation of proteins from the venom of the Indian cobra snake (Naja naja). Published in the Journal of Chromatography Open, the work presents an online 2D workflow that combines hydrophobic interaction chromatography (HIC) in the first dimension and reversed-phase LC (RPLC) in the second (1).

A Majestic Cobra Displays Its Hood In Dark Surroundings © Hasna90 - stock.adobe.com

The Indian cobra is one of the most venomous snakes in India and is known for its diverse and potent toxin profile. If bitten, the venom primarily affects the nervous system, causing muscle paralysis; in severe cases, it can lead to cardiac arrest or respiratory failure. The venom’s proteinaceous complexity—ranging from 5 to 200 kDa—can pose challenges for complete characterization. While methods such as RPLC (2), size-exclusion chromatography (SEC) (3), and ion-exchange chromatography (IEX) (4) have seen widespread use in venom analysis, their use in a single chromatographic step often limits a study’s findings (1).

The team therefore designed a native 2D-LC workflow, with HIC used in the first dimension to separate proteins under non-denaturing conditions based on hydrophobicity. Selected fractions or the entire eluate were then desalted and further resolved by short-gradient RPLC in the second dimension, with subsequent high-resolution mass spectrometric detection.

A standalone RPLC–MS method identified 35 distinct proteins in Indian cobra venom—primarily low-mass three-finger toxins (3FTxs) (small non-enzymatic proteins that affect the nervous system), phospholipases A₂ (PLA₂s) (a significant component that breaks down cell membranes), and snake venom metalloproteinases (SVMPs) (responsible for hemorrhage and facilitating blood loss) (5). Meanwhile, a standalone HIC method yielded 30 protein peaks. In contrast, the heart-cut 2D approach enabled the identification of 116 proteins, while the comprehensive mode revealed 134 proteins. Of these, 81 were uniquely detected via the comprehensive 2D approach, while 18 were identified with the heart-cut method (1). An earlier study reported a maximum of 115 proteins from Indian cobra venoms using various offline and denaturing workflows (6).

The workflow’s sample-to-sample reproducibility was tested, with %RSD values below 10% for all detected peaks, even in triplicate runs. The method’s 45-min run time means that drug candidates can be screened against individual venom components. The authors highlighted that, unlike RPLC, which often denatures proteins irreversibly, the HIC-RPLC–MS method captures biologically active conformations.

While the method demonstrated good selectivity and robustness at a 10-μg injection level, sensitivity at very low injection levels (5 µg) remains an area requiring improvement. The authors suggest future optimization to accommodate ultra-trace detection. Furthermore, while the method demonstrates high selectivity, further investigation into potential matrix effects and the detection of low-abundance proteins is necessary to ensure its broader applicability across diverse venom samples (1).

References

(1) Kumar, S.; Krishna K.; Rathore, A. S. Advanced Two-Dimensional Liquid Chromatography Workflow for Enhanced Resolution of Protein Components in Indian Cobra (Naja naja) Venom Using Hydrophobic Interaction-Reverse Phase Chromatography Coupled with Mass Spectrometry. J. Chromatogr. Open 2025, 7, 100211. DOI: 10.1016/j.jcoa.2025.100211

(2) Dutta, S.; Chanda, A.; Kalita, B.; et al. Proteomic Analysis to Unravel the Complex Venom Proteome of Eastern India Naja Naja: Correlation of Venom Composition with its Biochemical and Pharmacological Properties. J. Proteomics 2017, 156, 29–39. DOI: 10.1016/j.jprot.2016.12.018

(3) Terzioglu, S.; Bittenbinder, M. A.; Slagboom, J.; et al. Analytical Size Exclusion Chromatography Coupled with Mass Spectrometry in Parallel with High-Throughput Venomics and Bioassaying for Venom Profiling. Toxins (Basel) 2023, 15, 552. DOI: 10.3390/toxins15090552

(4) Bordon, K. C. F.; Wiezel, G. A.; Cabral, H.; Arantes, E. C. Bordonein-L, A New L-amino Acid Oxidase from Crotalus durissus terrificus Snake Venom: Isolation, Preliminary Characterization and Enzyme Stability. J. Venom Anim. Toxins Incl. Top. Dis. 2015, 21, 26. DOI: 10.1186/s40409-015-0025-8

(5) Takeda, S.; Takeya, H.; Iwanaga, S. Snake Venom Metalloproteinases: Structure, Function and Relevance to the Mammalian ADAM/ADAMTS Family Proteins. BBA Proteins and Proteomics 2012, 1834, 164–176. DOI: 10.1016/j.bbapap.2011.04.009

(6) Vanuopadath, M.; Raveendran, D.; Gopalakrishnan Nair, B.; Sadasivan Nair, S. Venomics and Antivenomics of Indian Spectacled Cobra (Naja naja) from the Western Ghats. Acta Tropica 2022, 228, 106324. DOI: 10.1016/j.actatropica.2022.106324