ABSTRACT: Three flasks of asynchronous blood stage P. falciparum Dd2 culture were grown to ~15% parasitemia, 4% hematocrit using the Trager- Jensen method. Per biological replicate, parasites were treated with 5 x EC50 or EC90 of compound depending on the slope of the EC50 curve for 3 hours. Compounds are DC-12717, DC-12834, BI-2536, GSK461364, and DC-12862. All compounds have three biological replicates, except DC-12862 which has one. As a control, culture was treated with an equal volume of DMSO vehicle. Parasites were collected through saponin lysis of erythrocytes, and parasite pellets were resuspended in 8 M urea lysis buffer (8 M urea, 50 mM Tris pH 8.5, 1% SDS), supplemented with a protease and phosphatase inhibitor cocktail. Parasites were lysed by sonication, and protein was quantified with the BCA protein assay. Samples were reduced, alkylated, resuspended, and digested. Resulting peptides were quantified and labelled with isobaric tandem mass tags (TMT). Following a ratio check, phospho-enrichment was performed with a Pierce High-Select Fe-NTA Phosphopeptide Enrichment Kit. The resultant phosphopeptide eluate was analyzed with LC-MS2. The peptide flowthrough was fractionated by basic reverse phase HPLC and similarly analyzed with LC-MS3. Spectra were searched against a composite P. falciparum and human Uniprot database (https://www.uniprot.org/), using the COMET algorithm. Peptide matches were filtered with a 1% false discovery rate (FDR), and peptides with a summed signal-to-noise (SN) threshold of >100 were quantified. For phosphopeptides, a 1% FDR filter was used, and a summed SN threshold of greater than or equal to 100.
Channels:
126 - Culture 1 Control,
127N - Culture 2 Control,
127C - Culture 3 Control,
128N - Culture 1 DC-12717
128C - Culture 2 DC-12717
129N - Culture 3 DC-12717,
129C - Culture 1 DC-12835,
130N - Culture 2 DC-12835,
130C - Culture 3 DC-12835,
131N - Culture 1 BI-2536,
131C - Culture 2 BI-2536,
132N - Culture 3 BI-2536,
132C - Culture 1 GSK461364,
133N - Culture 2 GSK461364,
133C - Culture 3 GSK461364,
134N - Culture 1 DC-12862
Debopam Chakrabarti 
