ABSTRACTThe flagellated kinetoplastid protozoan and causative agent of human Chagas disease,Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments,T. cruziuses its single flagellum to propel motile life stages and, in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of theT. cruziflagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase TurboID to the flagellum or cytosol in replicative stages ofT. cruzito identify proteins that are enriched in the flagellum by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins inT. cruziepimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these enriched flagellar proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to theT. cruzilineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments inT. cruzi. The proteomic data sets generated in this work offer a valuable resource to facilitate functional investigation of the understudiedT. cruziflagellum.IMPORTANCETrypanosoma cruziis a protozoan parasite that causes Chagas disease, which causes substantial morbidity and mortality in South and Central America. Throughout its life cycle,T. cruziinteracts with insect and mammalian hosts via its single flagellum, establishing intimate contact with host membranes. Currently, few flagellar proteins have been identified inT. cruzithat could provide insight into the mechanisms involved in mediating physical and biochemical interactions with the host. Here, we set out to identify flagellar proteins in the main replicative stages ofT. cruziusing a proximity-labeling approach coupled with mass spectrometry. The >200 candidate flagellar proteins identified represent the first large-scale identification of candidate flagellar proteins inT. cruziwith preliminary validation. These data offer new avenues to investigate the biology ofT. cruzi-host interactions, a promising area for development of new strategies aimed at the control of this pathogen.