The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of
substrate proteins with myristic acid in eukaryotes and is a validated drug target in the
parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping
sickness). N-Myristoylation typically mediates membrane localisation of proteins and is
essential to the function of many. However, only a handful of proteins are experimentally
validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an
alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue
to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we
combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical
proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream
form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins,
phosphatases and many uncharacterized proteins as substrates of NMT in the parasite,
providing a global view of the scope of this important protein modification and further
evidence for the crucial and pleiotropic role of NMT in the cell.