Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Camila Takeno Cologna, Renata Santos Rodrigues, Jean Santos, Edwin de Pauw, Eliane Candiani Arantes, Loïc Quinton

Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom fromNeoponeraants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from severalNeoponeraspecies have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis ofNeoponera villosavenom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Specimens ofN. villosaants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 800-4000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family. In addition, smaller fragments related to ponericins were also identified, suggesting that this class of antimicrobial peptide might undergo enzymatic cleavages. There are substantial differences among the venom ofN. villosaants collected in different seasons and from different nest habitats. The venom composition is affected by climate changes that influence prey availability and predator presence. Clearly, nano-LC-MS boosted the knowledge about ant venom, a rich source of unexplored and promising bioactive compounds.