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

• Analysis of Naja naja venom proteins through -two-dimensional approach • Combination of hydrophobic interaction and reverse-phase chromatography was used • Standalone reverse-phase chromatography identified only 35 distinct proteins • Stand-alone hydrophobic interaction chromatography exhibited 30 distinct proteins • Proposed method identified a total of 134 unique proteins in the venom Snake venom exhibits complexity due to its composition of a diverse array of proteins and peptides, ranging in molecular masses from approximately 5 kDa to 200 kDa. A comprehensive understanding of the venom proteome is imperative for the design of effective therapeutic candidates. Conventional methodologies often fall short of achieving comprehensive separation of venom proteins. In the current study, we introduced a novel, online two-dimensional liquid chromatography coupled with mass spectrometry for the separation of proteins within the venom of the Indian cobra snake ( Naja naja ). The proposed methodology utilizes hydrophobic interaction chromatography as the first dimension and reversed-phase liquid chromatography coupled with mass spectrometry as the second dimension. Utilizing the proposed two-dimensional workflow, we successfully identified 116 proteins with the heart-cut method and 134 proteins with the comprehensive method from Naja naja venom. Notably, 18 proteins were exclusively identified with the heart-cut method, while 81 were unique to the comprehensive approach. In contrast, a standalone reverse-phase chromatography coupled with mass spectrometry method identified only 35 distinct proteins, whereas the stand-alone hydrophobic interaction chromatography method exhibited 30 distinct protein peaks. The proposed strategy offers advantages including speed (a sample run time of 45 minutes), detailed protein identification, and preservation of protein biological activity, particularly notable when assessing the interactions of anti-venom agents with venom components.