In-depth proteomic analysis of Plasmodium berghei sporozoites using trapped ion mobility spectrometry with parallel accumulation-serial fragmentation

Malaria is caused by Plasmodium spp. protozoan parasites, which are transmitted by female anopheline mosquitoes in the form of sporozoites. Once deposited in the dermis during the blood meal of the mosquito, sporozoites rapidly migrate to the liver for an initial and obligatory round of replication inside hepatocytes, before exponential multiplication of the parasite in the blood and onset of the malaria disease. Sporozoites and liver stages provide attractive targets for the development of a malaria vaccine. Until now, a single antigen from Plasmodium falciparum, the deadliest species infecting humans, has been considered for subunit vaccine clinical development, with limited success so far. This emphazises the need to identify novel targets. In this context, defining the parasite proteome is important not only to guide the down-selection of potential candidate antigens, but also to allow a better understanding of the parasite biology. Previous studies have determined the total proteome of sporozoite stages from the two main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, a parasite that infects rodents. Another murine malaria parasite, P. berghei, has been widely used to investigate the biology of Plasmodium pre-erythrocytic stages. However, a deep view of the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage of a novel highly sensitive timsTOF PRO mass spectrometer, based on trapped ion mobility spectrometry with parallel accumulation-serial fragmentation. Combined with three alternative methods for sporozoite purification, this approach allowed us to identify the deep proteome of P. berghei sporozoites using low numbers of parasites. This study provides a reference proteome for P. berghei sporozoites, identifying a core set of proteins expressed accross species, and illustrates how the unprecedented sensitivity of the timsTOF PRO system enables deep proteomic analysis from limited sample amounts.