A post-translational modification perspective of Bothrops venom toxins: sitespecific proteomic characterization of N-glycans and Cys residues

• Ano de defesa: 2021
• Autor(a) principal: Silva, Débora Andrade
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Espectrometria de massas, Proteômica; Mass spectrometry; N-glicosilação de proteínas; Protein N-glycosylation; Proteomics; Snake venom; Veneno de serpente
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/46/46131/tde-10042025-110422/

Snake venoms are complex mixtures of proteins and peptides, used for predation and defense, which appeared during the development of the order Squamata and later evolved in a variable mode in the group of advanced snakes. The variability of their compositions can be observed in all taxonomical levels, however, the factors that drove and selected such features are still under discussion. Post-translational modification (PTM) is one of the mechanisms involved in the generation of venom protein proteoforms. The most known PTMs are protein glycosylation, oligomerization of polypeptide chains, proteolytic processing, and disulfide bond formation. In this study we identified N-glycosylation sites and Cys-containing peptides in venom proteins of the Bothrops genus (B. alcatraz, B. cotiara, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, and B. moojeni) using mass spectrometry. In chapter 1, the analysis of N-glycosylation in proteins was performed by proteolysis with trypsin allied to glycopeptide enrichment using TiO2 and hydrophilic interaction chromatography. Glycopeptide enriched fractions were split in two parts: (i) for enzymatic de-N-glycosylation and analysis by LC-MS/MS, and (ii) for direct analysis of intact N-glycosylated peptides by LC-MS/MS. The results revealed the occurrence of a high-level of heterogeneity in N-glycosylation site occupancy and confirmed the different N-glycan repertoire of Bothrops venom proteins. In chapter 2, we explored the presence of free Cys residues in venom proteins and performed the relative quantification using tandem mass tag (TMT) labeling. The venom proteins were initially labeled with N-ethylmaleimide, and after proteolysis with trypsin, the peptide mixtures were labeled with different TMTs, combined and the Cys-containing peptides were enriched using thiol-Sepharose. The resin non-bound and bound fractions were fractionated by high pH reversed-phase chromatography and submitted to LC-MS/MS analysis. The results revealed the presence of free Cys residues in protein positions that involve disulfide bond formation, indicating that there could be more than one disulfide bond pattern occurring in venom proteins. Further, the relative protein quantification showed differential abundance in most toxin classes, and a hierarchical cluster analysis classified the seven venoms in three clusters, in a pattern similar to that observed in the phylogeny of these snakes.