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Ectatomma tuberculatum has one of the most toxic venoms known among ants but there is no detailed study on its characteristics. In light of this, knowing the venom’s chemical composition is of paramount importance in order to obtain information about the mechanisms of its components. Several bioactive molecules have already been identified in Hymenoptera venoms, i.e., proteins such as phospholipases, hyaluronidases, and proteinases, as well as peptides. Protein databases show that information on protein components of ant venoms has been recently growing exponentially. In this study, we have identified proteins from the venom of Ectatomma tuberculatum by means of 2D PAGE, followed by tandem nanochromatography with mass spectrometry. A total of 48 proteins were identified, of which 42 are involved in metabolic processes, transport, and structural support. Moreover, six of them show similarity with not yet characterized proteins. Nine proteins are related to the attack/defense or maintenance process of the colony (colony asepsis, conservation of venom constituents, venom diffusion on prey, paralysis of prey, alteration of homeostasis, and cellular toxicity). Our findings may contribute to the identification of new natural prototypes of molecules to be synthesized and used in several areas of pharmacology. [ABSTRACT FROM AUTHOR]