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.

female anopheline mosquitoes in the form of sporozoites. Once deposited in the dermis 26 during the blood meal of the mosquito, sporozoites rapidly migrate to the liver for an 27 initial and obligatory round of replication inside hepatocytes, before exponential 28 multiplication of the parasite in the blood and onset of the malaria disease. Sporozoites 29 and liver stages provide attractive targets for the development of a malaria vaccine. Until 30 now, a single antigen from Plasmodium falciparum, the deadliest species infecting 31 humans, has been considered for subunit vaccine clinical development, with limited 32 success so far. This emphazises the need to identify novel targets. In this context, defining 33 the parasite proteome is important not only to guide the down-selection of potential 34 candidate antigens, but also to allow a better understanding of the parasite biology. 35 Previous studies have determined the total proteome of sporozoite stages from the two 36 main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, a parasite 37 that infects rodents. Another murine malaria parasite, P. berghei, has been widely used to 38 investigate the biology of Plasmodium pre-erythrocytic stages. However, a deep view of 39 the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage 40 of a novel highly sensitive timsTOF PRO mass spectrometer, based on trapped ion 41 mobility spectrometry with parallel accumulation-serial fragmentation. Combined with 42 three alternative methods for sporozoite purification, this approach allowed us to identify 43 the deep proteome of P. berghei sporozoites using low numbers of parasites. This study 44 provides a reference proteome for P. berghei sporozoites, identifying a core set of proteins 45 expressed accross species, and illustrates how the unprecedented sensitivity of the 46 timsTOF PRO system enables deep proteomic analysis from limited sample amounts. 47

Introduction
year [1], remains a major public health problem in many countries. Significant progress 51 has been achieved over the past decades in reducing malaria incidence and mortality, 52 through the systematic use of insecticide-treated bednets and potent antimalarial 53 artemisinin-based drug combinations. However, progress in malaria control has recently 54 stalled, and the continued emergence of parasite and mosquito resistance to antimalarial 55 medicines and insecticides, respectively, is a serious threat to malaria control. The disease 56 is caused by parasites of the genus Plasmodium, which are transmitted via the bite of 57 infected Anopheles female mosquitoes. Invasive forms called sporozoites present in the 58 salivary glands of the vector are deposited in the dermis during a blood meal. After 59 traversing the skin, motile sporozoites traffic to the liver through the blood stream, and 60 reach the liver parenchyma where they invade hepatocytes for an initial and obligatory 61 replication phase, resulting in the release of tens of thousands of merozoites [2]. These 62 merozoites invade erythrocytes and initiate the exponential asexual reproduction of the 63 parasite in erythrocytes, causing the symptomatic phase of malaria. Of the Plasmodium 64 species infecting humans, Plasmodium falciparum is the most prevalent and the deadliest 65 species, especially in subsaharan Africa. P. vivax, the second most important species in 66 humans, is widely distributed around the world but causes less severe malaria. One 67 particularity of P. vivax is to cause relapsing malaria episodes, due to hypnozoites, which 68 are dormant liver-stage parasites that can reactivate weeks or months after parasite 69 transmission by a mosquito. 70 Infection of the liver by sporozoites is an essential and clinically silent phase of the 71 malaria life cycle, and has long been considered as an ideal target for a malaria vaccine 72 Here, we took advantage of the highly sensitive timsTOF PRO mass spectrometer 111 to determine the total proteome of P. berghei sporozoites. This system is equipped with 112 the trapped ion mobility spectrometry (TIMS) technology for parallel accumulation serial 113 fragmentation (PASEF). The dual TIMS technology enables ions trapping in the front 114 section and ions separation according to their ion mobility in the rear section. Combined 115 with rapid quadrupole switching and TOF detector, the timsTOF PRO enables the 116 fragmentation of multiple simultaneously eluting precursor ions with a near 100% duty 117 cycle. The timsTOF PRO offers speed and sensitivity gains of up to 10-fold compared to 118 other mass spectrometry approaches [18]. Combined with three alternative methods for 119 sporozoite purification, this system allowed us to identify the deep proteome of P. berghei 120 sporozoites using unprecedented low numbers of parasites. 121 122 2. Results and discussion 123 infected mosquitoes, resulting in sample contamination with proteins from the mosquito 126 or its microbiota. Purification of sporozoites after dissection is a crucial step to reduce the 127 quantity of contaminating proteins of mosquito origin. We compared three different 128 methods for parasite purification: 1) the density gradient purification procedure, 129 developped by Kennedy et al [5], used as a reference method 2) immunocapture of 130 sporozoites using magnetic beads coupled to anti-CSP antibodies and 3) sorting of 131 fluorescent sporozoites by flow cytometry. Figure 1 illustrates the workflow that was 132 used for the proteomic analysis of P. berghei sporozoites. In total, 4 independent 133 sporozoite preparations were purified with the density gradient method, 2 using 134 magnetic beads and 4 by flow cytometry, as summarized in Table 1. Purification using the density gradient protocol was relatively easy to execute but 140 the sporozoite recovery rate was highly variable, ranging from 9 to 39% (mean 28%) 141 (Table 1). Despite this lack of reproducibility in our hands, the purification efficiency was 142 satisfying, with P. berghei proteins representing between 30 and 55% (mean 39.93%) of 143 the total number of proteins identified by mass spectrometry (Figure 2A), which is 144 consistent with values reported with other species [8]. Immunocapture of sporozoites 145 with anti-CSP antibodies coupled to magnetic beads was easy and rapid to execute. 146 However, mosquito debris remained abundant despite extensive bead washes. In 147 addition, as sporozoites were agglutinated with the beads after elution, it was impossible 7 to the determine the yield of recovered sporozoites with this method. Nevertheless, 149 samples were analyzed by mass spectrometry, resulting in around 35% of identified 150 proteins being of parasite origin ( Table 1 and Figure 2A). Sorting of GFP-expressing 151 sporozoites by flow cytometry also showed highly variable recovery rates, varying 152 between 10 and 55% ( Table 1). The poor recovery rate observed with some of the 153 samples is probably due to the intrinsic low efficiency of the sorting method and to the 154 fact that sorted parasites were highly diluted during the procedure, increasing the risk of 155 parasite loss during subsequent centrifugations. Nevertheless, the purification efficiency 156 was satisfying, with P. berghei proteins representing up to 60% of the total number of 157 proteins identified by mass spectrometry ( Table 1 and Figure 2A). Based on these 158 results, we conclude that both immunocapture and FACS sorting of sporozoites provide 159 valuable alternative to the density gradient reference method. 160 161

Effect of sporozoite sample size on proteome identification 162
We next analyzed the effect of sample size on the performance of the timsTOF PRO 163 system for protein identification, for the three purification methods. Remarkably, mass 164 spectrometry analysis of as few as 100,000-200,000 sporozoites purified by the density 165 gradient method (DG1 and DG3 samples in Table 1) identified 500-900 P. berghei 166 proteins ( Figure 2B), and a single analysis of 900,000 sporozoites (DG2 sample in Table  167 1) resulted in the identification of 1532 P. berghei proteins ( Figure 2B), which represents 168 93 % of the total number of proteins identified in the entire study (see below). 169 When combining all the identification results from 10 independent experiments (18 170 injections), we detected a total of 1648 proteins in P. berghei salivary gland sporozoites 171 (Table S1 and Table S2). Increasing the number of technical replicates (multiple injections 172 systems (59 to 76%) [19,20], and can probably be explained by the high frequency of the 175 selection/fragmentation cycle (up to 100 per cycle). In contrast, increasing the number of 176 biological independent samples increased substantially the number of proteins identified 177 in P. berghei sporozoites ( Figure 2D (Table S3). In contrast, 51 (3.0%) 194 proteins were identified only in sporozoites from the rodent malaria species (P. yoelii and 195 P. berghei) and 89 (5.4%) only in P. berghei. 196

Analysis of sporozoite protein families 198
We next scrutinized the P. berghei proteome dataset for a selection of protein 199 families, in comparison with published proteomes from P. falciparum, P. vivax and P. yoelii 200 (Table S3)

Concluding remarks 240
This study provides a reference proteome dataset for P. berghei sporozoites, 241 complementing previous proteomic studies of P. falciparum, P. vivax and P. yoelii 242 parasites. We identify a conserved set of more than 1200 sporozoite proteins shared 243 between the four species, and found some differences between the datasets. Further 244 investigations will be required to determine whether these differences reflect biological 245 specificities or merely variations in protein abundance. Finally, identification of the P.