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Protein N-myristoylation is catalysed by N-myristoyltransferase, an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescentlabelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of labelfree and SILAC-based proteomic strategies in the presence and absence of the NMT inhibitor DDD856464, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43-0.46% of the proteome is Nmyristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5-1.7%).