Functionally mapping the diversification of African trypanosomes using spatial proteomics Nicola Moloney, Konstantin Barylyuk, Kathryn Lilley, Ross Waller, Paula MacGregor Department of Biochemistry, University of Cambridge African trypanosomes are unicellular parasites with a life cycle comprising of insect and vertebrate stages. They cause disease with devastating economic impact in sub-Saharan Africa through infection of livestock. Discrete phenotypic differences between species and between life-cycle stages are concomitant with the host and tissue tropisms observed within this group. Understanding the cell biology that drives these diversities requires a comprehensive functional map of each group member which, collectively, is currently lacking. Protein function is often intimately linked with localisation and as a consequence the subcellular distribution of a protein provides information on its role in the cell. We have optimised an effective method for resolving subcellular compartments in Trypanosoma brucei and Trypanosoma congolense and implemented it in the spatial proteomics strategy of hyperLOPIT (hyperplexed localisation of organelle proteins by isotope tagging). Between the insect and vertebrate stages, represented by the procyclic and bloodstream forms respectively, we have detected 7246 proteins in T. brucei and 7133 in T. congolense. Of these proteins, 6146 T. brucei proteins (n = 3) and 7058 T. congolense proteins (n = 1) are sufficiently quantified for inclusion in spatial proteome characterisation. Unsupervised clustering indicates approximately 20 subcellular niches are differentially resolved in each organism. Preliminary classification of uncharacterised proteins in T. brucei using the TAGM-MAP (t-augmented Gaussian mixture maximum a posteriori) model localised 3698 in bloodstream form and 4234 in procyclic form. This work provides a comprehensive map of T. brucei and T. congolense spatial proteomes. Individually, these data sets will guide the determination of uncharacterised protein functions, particularly for T. congolense, where high-throughput functional analysis lags behind that of T. brucei. Further, comparative analysis of these maps will yield insight into the evolutionary diversification of these species and the effects of specialisation on the molecular biology and subcellular architecture of the parasite cell.