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Fig. 2 | Military Medical Research

Fig. 2

From: Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA

Fig. 2

Identification of CQ target proteins by CQP through ABPP. a General workflow of photoaffinity CQP probe-mediated ABPP used to label and identify CQ target proteins. Parasite lysates were incubated with CQP probe, CQ or vehicle followed by irradiation with UV light (λ = 365 nm). The alkyne tag on the probe allows for the subsequent attachment of a fluorescent tag or biotin moiety through CuAAC-mediated click chemical reaction. The fluorescence labeling of parasite proteins is visualized by fluorescence scanning after resolving on SDS-PAGE gel. The pull-down of CQP-labeled proteins by biotin-streptavidin affinity purification is subject to TMT-based labeling and quantification on LC–MS/MS. The chemistry structures for fluorescent and biotin tags seen in Additional file 2: Fig. S1. b Determination of antimalarial activity of CQP and CQ in P. falciparum 3D7 strain. c In situ labeling of CQP in parasites living in infected red blood cells. The parasites were released and the lysates were prepared for click reaction. The amounts of proteins being labeled were in a dose-dependent manner after UV irradiation, while there was no labeling without UV irradiation. d In vitro labelling of CQP in extracted parasite lysates were consistent with the in situ labeling as shown in c. e Labeling of CQP-target proteins in parasite lysate can be competed with excess CQ. f Confocal microscopy showing the distribution of CQP (2 µmol/L) inside the P. falciparum 3D7 parasites, and eliminated mostly with excess CQ (20 µmol/L). g Scatter plot of 60 quantified proteins from CQP ABPP experiment. Each dot represents a quantified protein, with the x-axis represents the mean of log2 protein abundance difference between CQP and DMSO control group, while y-axis represents the mean of log2 difference between CQP and CQ + CQP group. Two biological replicates were included in the experiment. The dashed line represents the fold change cutoff criteria (> 1.2) used for hit selection. The gray points represent proteins with Padj > 0.05. Padj were generated from the empirical Bayes test model and adjusted using Benjamini–Hochberg. The proteins measured with only 1 peptide spectrum match were regarded as low-confidence, and colored in orange. The remaining 40 proteins were kept as high-confidence targets and colored in red and the dot size is proportional to the associated PSM numbers. DMSO dimethyl sulfoxide, CQ chloroquine, CQP chloroquine analog probe, TAMRA carboxytetramethylrhodamine, CuAAC copper-catalyzed azide-alkyne-cycloaddition, SDS-PAGE sodium lauryl sulfate–polyacrylamide gel electrophoresis, TMT tandem Mass Tag, LC–MS/MS liquid chromatography-tandem mass spectrometry, Coo comassie, DAPI 4',6-diamidino-2-phenylindole, HZ hemozoin, RBC red blood cell, FC fold change, PSMs peptide-spectrum matches

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