DOI: 10.5061/dryad.kwh70rz9s
title: Proteome of peripheral mononuclear cells (PBMCs) from asymptomatic malaria and uninfected individuals and the ensuing malaria episodes

description: Cumulative malaria parasite exposure in endemic regions often results in
 the acquisition of partial immunity and asymptomatic infections. There is
 limited information on how host-parasite interactions mediate maintenance
 of chronic symptomless infections that sustain malaria transmission. Here,
 we have determined the gene expression profiles of the parasite population
 and the corresponding host peripheral blood mononuclear cells (PBMCs) from
 21 children (&lt;15 years). We compared children who were defined as
 uninfected, asymptomatic and those with febrile malaria. Children with
 asymptomatic infections had a parasite transcriptional profile
 characterized by a bias toward trophozoite stage (~12 hours-post invasion)
 parasites and low parasite levels, while earlier ring stage parasites were
 characteristic of febrile malaria. The host response of asymptomatic
 children was characterized by downregulated transcription of genes
 associated with inflammatory responses, compared to children with febrile
 malaria. Interestingly, the host responses during febrile infections that
 followed an asymptomatic infection featured stronger inflammatory
 responses, whereas the febrile host responses from previously uninfected
 children featured increased humoral immune responses. The priming effect
 of prior asymptomatic infection may explain the blunted acquisition of
 antibody responses seen to malaria antigens following natural exposure or
 vaccination in malaria endemic areas.
description: Proteins were extracted from PBMCs by resuspending the pellet
 with 5µl of 6M UREA (Thermo scientific). The protein samples were then
 adjusted with 50mM Triethylamonium bicarbonate (TEAB, Sigma-Aldrich) to
 100µl and the protein concentration determined using the Bicinchoninic
 acid (BCA) protein assay (Thermo scientific). The protein samples were
 then reduced with 40mM dithiothretol, alkylated with 80mM iodoacetamide in
 the dark, and quenched with 80mM iodoacetamide at room temperature,
 followed by digestion with1µg/µl of trypsin (57). Nine pools, each
 containing 9 samples and 1 control for batch correction, were prepared by
 combining 1µl aliquots from each sample. The samples were pooled using a
 custom randomization R script. The pooled samples were then individually
 labelled using the Tandem Mass Tag (TMT) 10-plex kit (Thermo Scientific)
 according to the manufacturer’s instructions. One isobaric tag was used
 solely for the pooled samples and combined with peptides samples labelled
 with the remaining 9 tags. The labelled peptide pools were then desalted
 using a P10 C18 pipette ZipTips (Millipore) according to the
 manufacturer’s instructions. Eluted peptides were dried in a Speedvac
 concentrator (Thermo Scientific) and re-suspended in 15μl loading solvent
 (98% H2O, 2% acetonitrile, 0.05% formic acid). The peptides were then
 quantified using Qubit Protein Assay Kit (Thermo Fisher Scientific). A
 standardized protein concentration of 5µg was finally injected into the
 LC-MS/MS for analysis. The peptides were then loaded onto the liquid
 chromatography and separated on reverse-phase analytical column of 75µm x 50cm C18 (Thermo Scientific) and
 measured using a Q Exactive Orbitrap mass spectrometer as described by
 (Njunge <em>et al</em>., 2019) and mass spectrometer output
 files generated. To identify and quantify proteins,
 mass spectrometer output files were analyzed using MaxQuant software
 version 2.0.3.0 (Cox and Mann, 2008) by searching against the Uniprot
 human proteome (downloaded on 10/06/2021) using the Andromeda search
 engine (Cox<em> et al</em>., 2011). N-terminal acetylation and
 methionine oxidations were set as variable modifications while cysteine
 carbamidomethylation and TMT-10plex labelled N-terminus and lysine were
 set as a fixed modification. The false discovery rate (FDR) cut-off was
 set as 0.01 for both proteins and peptides with a minimum length of seven
 amino acids and was determined by searching a reverse database. Enzyme
 specificity was set as C-terminal to arginine and lysine with trypsin as
 the protease. Only up to two missed cleavages were allowed in the database
 search. Peptide identification was performed with an allowed fragment mass
 deviation maximum of 20 ppm (parts per million) and an initial precursor
 mass deviation maximum of 7 ppm. Default parameters for Orbitrap-type data
 were used. The pooled sample channels were used for batch correction. The
 10-plex corrected reported ion intensity matrix was extracted from the
 protein groups output file and used for downstream analysis. Proteins
 matching the reversed part of a decoy database, potential contaminants and
 proteins only identified by a modification site, were excluded.
 Differential protein abundance analysis of the labelled samples
 intensities generated by MaxQuant were performed using PERSEUS v2.05.0
 MaxQuant software (MaxPlanck Institute of Biochemistry, Martinsried,
 Germany) as described in (Tyanova and Cox, 2018).
description: The files can be opened using MaxQuant software, specifically
 version 2.0.3.0 was used for analysis. Differential
 protein abundance analysis of MaxQuant output was done using PERSEUS
 version 2.05.0 software. Protein-protein interaction
 and Gene ontology analyses was perforened using STRING database version
 11.5 (https://string-db.org/).
description: # Proteome of peripheral mononuclear cells (PBMCs) from asymptomatic
 malaria and uninfected individuals and the ensuing febrile malaria
 episodes Proteins were extracted from peripheral mononuclear cells
 (PBMCs), pooled using Tandem Mass Tags (TMT) (10-plex) and injected into
 the LC-MS/MS for proteomics analysis. The output raw files were loaded
 into MaxQuant software v2.0.3.0 for protein quantification. The output
 from MaxQuant was then read using PERSEUS software v2.05.0 and
 differential protein abundance analysis performed. The Proteomics_metadata
 file contains the metadata that links each sample to the raw data files
 and the treatment group (condition). ## Description of the data and file
 structure The RAW data files provided contains the output data from the
 LC-MS/MS per each pool. The pools serve as the input data for MaxQuant
 software. The Proteomics_metadata contains the metadata information that
 links each sample to the condition/treatment group (i.e. asymptomatic,
 uninfected or febrile). * Code represents the short code given to each
 samples during analysis. * Experiment is the TMT batch/pool that
 correspond to the raw data file batches. * Condition refers to the
 treatment group that each sample belongs to. Asymptomatic and Uninfected
 refers to samples collected from individuals who had asymptomatic malaria
 infections or from uninfected individuals, respectively. The
 Febrile_Asymptomatic refers to samples collected when asymptomatic
 individuals presented with malaria symptoms. Febrile_Uninfected refers to
 samples collected when the uninfected individuals presented with malaria
 symptoms.