Proteomic landscapes of Pachycondila villosa ant venom by nano-scale chromatography and high resolution mass spectrometry

Introduction: It is estimated that the total number of molecules present in animal venoms is a collection of 40 million different compounds and, despite the efforts made, less than 0,01% of those compounds was identified and characterized to date. However, recent progresses in proteomic, in parallel with the advances of mass spectrometry have contributed to the study of those bio-libraries. The sensitivity improvement of these instruments allows the study of minimal amounts of sample still yielding a wealth of information. The present work aimed to perform a deep proteomic analysis of the venom from the ant Pachycondilyla villosa focusing on the de novo sequencing and the characterization of post translational modifications using high resolution mass spectrometers. Methods: The crude venom (0,5 ug) of P. villosa ants collected on Panga Natural Reserve (Uberlandia-Minas Gerais- Brazil) was diluted in 0,2% of formic acid and injected into a nanoACQUITY ULPC equipped with a monolithic PepSwift Capillary column 100µm x 25, hyphened to a Q Exactive Orbitrap mass spectrometer. The elution of the compounds was performed with a gradient of 3 to 50% of solution B in 80 minutes (A: H2O/FA 0.1%; B: ACN) at flow rate of 1 µL/min. All mass spectrometry analyses were performed in data dependent analysis (DDA) mode that automatically triggers the MS/MS experiments. The top 10 most intense peaks of each MS scan was fragmented by high-energy dissociation (HCD) and their corresponding MS/MS spectra were acquired. Preliminary data: Animal venoms are considered a rich source of biologically active compounds, which has been constantly selected and refined by the processes of natural evolution, in which each molecule is endowed with pharmacological properties highly valuable for scientific purposes. Despite the commitment, the exploration of these bio-libraries remains limited which might be related to the technological limitations that prevent full-scale investigation of these venoms. In addition, the conventional methods used to explore animal venoms are still time-consuming and require large amounts of samples, which restrict the studies for a few species. Unquestionably, the advances of proteomics and mass spectrometry instrumentations benefited a great deal the research on hymenoptera venom. Mostly due to their small size and therefore scarcely collected venom, this order has always been neglected and considered unfeasible to be studied through the known strategies. The present work represents the first report concerning the venom composition of P. villosa ant. The preliminary results already highlight the complexity of this venom, which showed to be composed by over 5000 different molecules. Most of those components fall into the 800- 4000 Da range, which is in agreement with other studies regarding ant´s venom composition. Most of the proteomics studies concerning ant venoms already revealed the presence of linear peptides below 5000 Da as major components. Those small peptides usually display antimicrobial activity and some of them hold additional insecticidal activity. Novel aspect: The results obtained already point out the biotechnological potential of P. villosa venom and highlight’s its complexit