A comparison of two different analytical workflows to determine the venom proteome composition of Naja kaouthia from North-East India and immunological profiling of venom against commercial antivenoms

Abstract

The Indian monocled cobra (Naja kaouthia) is one of the most prevalent venomous snakes in northeast India (NEI) and is the cause of many fatalities. The composition of NEI N. kaouthia venom (NkV) was deciphered using two different proteomic approaches: (i) 1D SDS-PAGE coupled to label-free quantification of protein bands using stringent identification criteria and (ii) reversed-phase high-performance liquid chromatography (RP-HPLC) followed by quantification based on area under the RP-HPLC peaks. The proteomic data from both strategies were compared. Proteomic analyses from both workflows identified 32 proteins (toxins) distributed over 10–14 snake venom protein families in NEI NkV. The relative abundances of the venom proteins determined from the analytical workflows coincided with the densitometry band intensities of the NEI NkV. Phospholipase A2 (13.1–16.0%) and three-finger toxins (58.5–64.2%) represented the most abundant enzymatic and non-enzymatic proteins in NEI NkV, respectively. Immuno-cross-reactivity studies by enzyme-linked immunoassay and immunoblot analyses pointed to the poor efficacy of commercial PAVs in recognizing the low molecular mass (<15 kDa) toxins of NEI NkV. Spectrofluorometric titration determined the presence of NEI NkV-specific antibodies in commercial PAV, at a level that was higher than that previously reported for eastern India NkV-specific antibodies in commercial antivenom.