Project description:Both single cell and bulk RNA sequencing was performed on expanding or differentiating snake venom gland organoids (from Aspidelaps Lubricus Cowlesi and Naja Nivea), or tissue (Aspidelaps Lubricus Cowlesi). Bulk RNA sequencing from the snake venom gland, liver and pancreas was performed to construct a de novo transcriptome using Trinity.
Project description:Three main top-down mass spectrometry methodologies were employed in a proof-of-concept study to characterise selected three finger toxins and phospholipase A2 toxins from the forest cobra (Naja melanoleuca) venom. The novel utility of data-independent acquisition, alternative hybrid ECD-CID fragmentation and the incorporation of cyclic ion mobility separation in a top-down proteomic workflow were explored for snake venom proteins.
Project description:Top-down proteomics of venom protein of venom-gland organoids aspidelaps. Samples were extracted with MilliQ water and proteins reduced with TCEP before top-down LC-MS/MS analysis.
Project description:Top-down proteomics of venom protein of venom-gland organoids aspidelaps. Samples were extracted with MilliQ water and proteins reduced with TCEP before top-down LC-MS/MS analysis.
Run 2
Project description:Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (LAAO, ~130 kDa) and the multi-chain toxin cobra venom factor (~147 kDa). The LAAO complex exhibited two clusters of multi-proteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new anti-venoms or other biotherapeutics.
Project description:The genus Bothrops is responsible for most part of envenomation accidents in Brazil. Bothrops pubescens is an endemic and neglected species in the Brazilian Pampa Biome. The characterization of its venom is essential since there is no data about it and can be helpful in the discovery of active biomolecules and for a better understanding of its action. We used top-down (TDP), native top-down, and bottom-up proteomic (BUP) approaches to characterize the venom of B. pubescens. We were able to identify 89 protein groups with the BUP approach and 40 unique proteoforms with the TDP approach, demonstrating the similarities and peculiarities of B. pubescens venom. We also identified a dimeric L-amino acid oxidase with using native TDP. Here we present for the first time a bothropic venom characterization through TDP approaches.
Project description:Venom proteomics analysis of Walterinnesia aegyptia and Walterinnesia morgani. For top-down analysis, venom samples were reduced with TCEP and measured via HPLC-MS/MS (Q-Exactive and LTQ-Orbitrap XL).