A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum–infected red blood cell proteome

Abstract

Background Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry–based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility. Results Here, we present a comprehensive P. falciparum–infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites. Conclusion A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway

“…Although this result is consistent with the dysregulated peptides being predominantly hemoglobin-derived, the statistical significance of the effect is difficult to assess. To this end, we repeated this analysis for each of the ~4700 proteins identified in our recent comprehensive proteomic analysis of P. falciparum -infected erythrocytes and quantified the number of peptide matches to each protein and then divided by protein length to yield a normalized estimate of the similarity of each protein to our significantly dysregulated peptides ( Siddiqui et al, 2022 ). By this measure, hemoglobin chains α, α2, β, and δ are four of the five most-similar proteins ( Figure 9C ).…”

“…falciparum and 1617 H . sapiens proteins that we recently identified in P. falciparum -infected erythrocytes ( Siddiqui et al, 2022 ).…”

“…LC-MS/MS was carried out using data-independent acquisition mode as described previously ( Siddiqui et al, 2022 ). Raw files were then processed using SpectronautTM 13.0 against an in-house generated P. falciparum ( Pf 3D7 line) spectral library as described previously ( Siddiqui et al, 2022 ).…”

“…Raw files were then processed using SpectronautTM 13.0 against an in-house generated P. falciparum ( Pf 3D7 line) spectral library as described previously ( Siddiqui et al, 2022 ). Following protein identification using the software SpectronautTM 13.0 as previously described ( Siddiqui et al, 2022 ), proteins identified were exported as an excel sheet and their fold change of drug-treated conditions relative to the 0 µM control for each experiment was calculated (only for proteins with intensities greater than 1x10 5 and with a minimum peptide count of 2), and the significance of the change was determined by a Welch’s t-test, with a p-value <0.05 deemed significant. Proteins that were significantly stabilized (fold change >1.15, p-value <0.05) at multiple concentrations of compound 3 were considered ‘hits’ and these were plotted using paired volcano plots.…”

Bio‐Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity

“…Mass Spectrometry Analysis: For MALDI-TOF MS/MS, the cells were lysed using a 1% sodium deoxycholate (SDC) solution in a 100 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 8.1) as previously described. [59] The lysate was heated at 95 °C for 10 min followed by probe sonication three times for 10 s to allow complete solubilization of membrane-bound proteins. The proteins were then reduced and alkylated using 40 mm of tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich, Australia) and 10 mm iodoacetamide (Sigma-Aldrich, Australia) respectively.…”