Analysis of the microheterogeneity in N- and O-glycosylation of snake toxins from Bothrops genus: identification of intact glycopeptides.

• Grant number: 18/08794-0
• Support type: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): August 06, 2018
• Effective date (End): February 05, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal researcher: Solange Maria de Toledo Serrano
• Grantee: Débora Andrade Silva
• Supervisor abroad: Martin R. Larsen
• Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Research place: University of Southern Denmark (SDU), Denmark
• Associated to the scholarship: 16/16935-7 - A post-translational modification perspective of Bothrops venom toxins: site-specific proteomic characterization of N-glycans and Cys residues., BP.DR

Abstract

Snake venoms are a rich source of molecules with biological activity, most of which are proteins. The presence of post-translational modification (PTM) in these proteins, such as glycosylation, has been evidenced in many studies. However, the full characterization of this PTM is still poorly known, regardless of its importance. Mass spectrometry is the primary technique for protein glycosylation analysis and the advances developed in the new spectrometers brought more possibilities to the challenge of the identification of glycosylation sites. In this type of analysis, simultaneous sequencing of the glycan chain and the peptide is essential. In this scenario, new fragmentation approaches, such as Higher Energy Collisional Dissociation (HCD) and HCD-product Dependent Electron-Transfer Dissociation (HCDpdETD) emerged as promising technologies. Thus, in this project, we propose to perform the analysis of the intact N-glycopeptides and O-glycopeptides present in glycoproteins from B. jararaca, B. alcatraz, B. insularis, B. cotiara, B. jararacussu, B. moojeni, and B. fonsecai venoms. As enrichment methodology of tryptic glycopeptides, we propose to use Hydrophilic Interaction Chromatography (HILIC) and titanium dioxide in combination and independent experiments. These data will allow for the understanding of the microheterogeneity of glycosylation in toxins, of the glycosylation pattern of each toxin class, and of possible additional glycan modifications, such as acetylation on sialic acid units. O-glycosylation is an especially exciting feature that deserves proper investigation, given the fact that its presence and importance in snake venom toxins remain fully uncharacterized.