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

Abstract

Sporozoites of the malaria parasite Plasmodium are transmitted by mosquitoes and infect the liver for an initial and obligatory round of replication, before exponential multiplication in the blood and onset of the disease. Sporozoites and liver stages provide attractive targets for malaria vaccines and prophylactic drugs. In this context, defining the parasite proteome is important to explore the parasite biology and to identify potential targets for antimalarial strategies. Previous studies have determined the total proteome of sporozoites from the two main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, which infects rodents. Another murine malaria parasite, P. berghei, is widely used to investigate the parasite biology. However, a deep view of the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage of the highly sensitive timsTOF PRO mass spectrometer, combined with three alternative methods for sporozoite purification, to identify the 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 across species, and illustrates how the unprecedented sensitivity of the timsTOF PRO system enables deep proteomic analysis from limited sample amounts.

Ion Mobility Coupled to a Time-of-Flight Mass Analyzer Combined With Fragment Intensity Predictions Improves Identification of Classical Bioactive Peptides and Small Open Reading Frame-Encoded Peptides

“…Also, the combination of trapped ion mobility spectrometry (tims) and Parallel Accumulation Serial Fragmentation (PASEF) effectively allows to maximize both throughput and sensitivity. This new technology has recently been used for analysis of complex samples in different research fields, such as the exploration of the proteome in human cerebrospinal fluid (Macron et al, 2020), detection of antibacterial peptides in the human endometrial fluid (Azkargorta et al, 2020) or the proteome of the malaria parasite Plasmodium (Hamada et al, 2021). To our knowledge, this is the first study where timsTOF is applied for neuropeptide and sORF discovery and only the second top-down MS study where non-digested peptide samples are identified using a timsTOF.…”

“…In this study, we introduce a two-step methodology combining firstly a trapped ion mobility spectrometry coupled to a time-of-flight mass analyzer (timsTOF) to generate the highest quality MSMS spectra, secondly the MS 2 ReScore (Silva et al, 2019) application leading to an improved identification of potential bioactive peptides in different regions of the mouse brain. timsTOF offers an enhanced peptide coverage and reduction in chemical noise (Lubeck et al, 2018) because of its extra dimension of separation (Meier et al, 2018), previously described in different applications in the field of clinical proteomics (Azkargorta et al, 2020;Macron et al, 2020;Hamada et al, 2021;Liu et al, 2021). The MS 2 ReScore tool, including MS 2 PIP that predicts the fragment ion intensity as an additional feature, DeepLC predicting retention times (Bouwmeester et al, 2021) and the post-processing tool Percolator, further boost the yield of identified peptides.…”

Research progress on biomarkers of pulmonary embolism

“…For DIA-MS, further innovations in instrumentation and informatics will be key to pushing the boundaries of improved sensitivity and throughput. [73][74][75][76]…”

Plasmodium sporozoites require the protein B9 to invade hepatocytes

“…In order to define the repertoire of 6-cys proteins expressed at the sporozoite stage, we first analysed the proteome datasets of P. falciparum 22,26 , P. vivax 24 , P. yoelii 26 and P. berghei 21 sporozoites. As expected, P36 and P52 were identified by mass spectrometry in sporozoites from all four species.…”

“…Plasmodium 6-cys proteins are typically expressed in a stage-specific manner, and have been implicated in protein-protein interactions in P. falciparum merozoites 16,17 , gametocytes 18,19 , ookinetes 20 and sporozoites 11 . Proteomic studies have shown that, in addition to P36 and P52, Plasmodium sporozoites express three 5 other 6-cys proteins, P12p, P38 and B9 [21][22][23][24] . While the contribution of P12p and P38 had not been studied until now, a previous study reported that the protein B9 is not expressed in sporozoites due to translational repression, and is not required for sporozoite invasion but is needed during infection of hepatocytes for early maintenance of the PV 15 .…”