Malaysia is home to various venomous snake species, including Ophiophagus hannah (OH), Naja sumatrana (NS), and Naja kaouthia (NK), which are medically significant due to their potent neurotoxins. Cobra venom primarily targets the neuromuscular junction, potentially causing respiratory arrest if untreated. The World Health Organization (WHO) recommends antivenom as the primary treatment, but its efficacy depends on venom composition. Therefore, understanding venom protein abundance is crucial for improving therapeutic strategies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard for venom proteomics, allowing detailed characterisation of venom complexity. Shotgun proteomics combined with label-free quantification (LFQ) has enhanced venom protein analysis, providing insights into toxin distribution and relative abundance. Despite its potential, peptide ion intensity, measured through peak intensity of specific m/z values, has not been previously applied in venom proteomics. This approach enables a direct correlation between ion concentration and protein abundance, offering improved venom composition estimation.
This study applied LFQ-based peptide ion intensity in PEAKS Studio (PEAKS 12.5, Bioinformatics Solutions Inc., Canada) to estimate relative venom protein abundance across OH, NS, and NK. To ensure sample consistency, venom protein content was first assessed using a bicinchoninic acid (BCA) assay before proteomic analysis. Using a standardised protein concentration of 150 µg/mL, 341 proteins were identified in OH, 147 proteins in NS, and 257 proteins in NK, surpassing previously reported numbers. The most abundant toxin families in OH were three-finger toxins (3FTxs) (42.4%), ohanin/vespryn (11.9%), and venom Kunitz-type proteins (8.4%). NS showed high abundances of 3FTxs (32.3%) and ohanin/vespryn (30.7%), while NK exhibited a balanced distribution, with ohanin/vespryn (24.1%) and 3FTxs (17.6%) as major components. By leveraging peptide ion intensity, this study provides deeper insights into venom variability, improving protein abundance estimation and supporting future antivenom development.