Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics

Abstract

The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

Keywords: Bothrops; Envenomation; L-amino acid Oxidase; Venomics.

Fig. 1.. Relative protein composition of B. pubescens venom.

The venom was analyzed by LC-MS/MS. Peptide search was performed on ProLuCID v1.3, and peptides were filtered by Search Engine Processor (SEPro). (A) Relative protein composition (%) of B. pubescens venom was estimated by label-free protein quantitation performed according to the normalized spectral abundance factor (NSAF). (B) Comparison between B. pubescens, B. moojeni (Melani et al., 2016), B. asper (Laemmli), B. jararaca (Mora-Obando et al., 2020), and B. diporus (Amorim et al., 2018) venom composition. SVMP - snake venom metalloproteinase, LAAO - L-aminoacidic oxidase, CTL - C-type lectin, PLA₂ - phospholipase A₂, SVSP - snake venom serine protease. Toxin families with less than 2% in the venom (others) include VEGF – vascular endothelial growth factor, BPP - bradykinin potentiating peptides, CRISP - cysteine-rich secretory protein, NGF - nerve growth factor, PDE - phosphodiesterase, GPC - glutamine-cyclotransferase, PLB - phospholipase B, NT - nucleotidases.

Fig. 2.. Identification of dimeric L-amino acid oxidase (LAAO) in B. pubescens venom.

(A) Chromatogram trace at 280 nm of the venom proteins highlighting the 13 fractions collected by size-exclusion chromatography (SEC). (B) Silver-stained SDS-PAGE analysis of the 13 SEC fractions. (C) Spectrum obtained using native top-down proteomics showing in red the charge state distribution of different LAAO multiproteoform complexes (MPC). (D) Deconvoluted MS1 spectrum and the intact mass of 8 different LAAO MPCs (a–h). The red squares indicate the fraction containing LAAO, and the red circle the LAAO bands region in the SDS-PAGE gel.