These proteins are mainly involved in processes such as DNA damage repair, protein synthesis and folding, and responses to oxidative stress. Proteome versus transcriptome analyses have been highly recommended for studies with tripanosomatids, as they have very peculiar molecular features concerning their gene expression control. As a kinetoplastid, T. cruzi transcription is polycistronic and gene regulation occurs mainly post-transcriptionally, with mature mRNAs being generated by trans-splicing and polyadenylation. The processing and stabilization of mRNAs are extremely important in trypanosomatid gene regulation. Furthermore, other dynamic control mechanisms, such as posttranslational modifications, are fundamental in the regulation of gene expression and need to be better characterized in these organisms. A time-course microarray study previously carried out by our group analyzed the T. cruzi gene expression in response to gamma radiation. Among the 273 differentially expressed genes, 160 were upregulated and 113 were downregulated. The majority of the genes with assigned functions was downregulated. Translation, protein metabolic processes, and the generation of precursor Butenafine hydrochloride metabolites and energy pathways were affected. Four mitochondrial genes and Retrotransposon Hot Spot genes were upregulated; likewise, the tyrosyl-DNA phosphodiesterase 1, a gene involved in DNA DSB repair, was also induced. Taking into account the T. cruzi gene expression peculiarities, analyses of proteome changes after irradiation in different time points may contribute to the understanding of the parasite response to such stress. In this work, we performed quantitative proteomic analyses using 2D-DIGE to ascertain the parasite response to ionizing irradiation. A total of 543 protein spots were found to be differentially expressed considering all analyzed time points and 53 different proteins were Penfluridol identified by tandem mass spectrometry. The great majority of the identified proteins was represented by several isoforms, suggesting that post-transcriptional and/or post-translational modifications are occurring as a consequence of gamma radiation exposure.