Supplementary Figure 2: General overview of proteome coverage in different ITDR sample preparation types. | Nature Protocols

Supplementary Figure 2: General overview of proteome coverage in different ITDR sample preparation types.

From: Cellular thermal shift assay for the identification of drug–target interactions in the Plasmodium falciparum proteome

Supplementary Figure 2

(a) Number of P. falciparum and non-hemoglobin H. sapiens proteins detected (PSM>3) across lysate (green), intact-cell (purple) and hemodepleted (‘Hb-‘) intact-cell (blue) ITDR 37 °C samples. Average values derived from 3 biological replicates are indicated on the graph with error bars representing Standard Deviation (SD). (b) Global hemoglobin content of Intact-cell ITDR 37 °C samples before and after the hemodepletion step, demonstrating successful removal of >90% of hemoglobin from the sample. Values indicated above each plot are an average of 8 biological replicates and error bars represent SD. Hemoglobin concentration was measured with Human Hb ELISA kit (Abcam, cat.no. ab 157707). (c) Peptide spectra match (PSM) counts for P. falciparum and non-hemoglobin H. sapiens proteins with PSM>3 detected in MS/MS analysis of three different sample preparation types: lysate (green), intact-cell (purple) and hemodepleted (‘Hb-‘) intact-cell (blue) ITDR 37 °C samples. Values are based on 3 biological replicates show significant increase in PSM in hemodepleted intact-cell samples. Error bars represent SD and p-values indicated on the plot were obtained from unpaired t-test. (d) Bland-Altman plot of PSM per protein difference in Hb- and non-depleted intact-cell sample preparation, demonstrating that hemodepletion results in higher average signal intensity for Plasmodium proteins identified in MS analysis (observed as the change in PSM number per protein in hemodepleted samples, relative to non-depleted samples). Plotted values are derived from overlapping proteins found in at least 1 biological replicate from each sample preparation type and possess PSM>3. (e) Venn diagram representing the overlap in proteins with PSM>3 identified between different sample preparation types. Data is based on confident protein identification in at least one out of three biological replicates of each sample type, demonstrating superior proteome coverage attained in lysate experiments and no bias in proteome coverage between intact-cell sample preparation variants. Figure is drawn based on unpublished data (experiments carried out by J.M.D., G.W. and K.D.).

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