Fig. 3. nHU-MS to survey affinities of the SNX27 PDZ domain proteome-wide.

(A) Volcano plot of the nHU-MS experiment performed with SNX27_PDZ bait on total Jurkat extracts (n = 6). Identified interaction partners with or without C-terminal putative PBMs are colored with orange or blue, respectively. (B) Volcano plot of the control pull-down experiment performed on the leftover resin of the nHU experiment (n = 6). Preys are colored according to their coloring on (A). (C and D) Converted affinities of nHU experiments measured with Jurkat (C) or SH-SY5Y (D) extracts. (A to D) P values were calculated using a two-sided unpaired t test for nHU-MS and AP-MS experiments. The statistical significance thresholds for binding (tan lines) were determined at 1 σ and P value < 0.05. (E) Correlation between apparent affinities of SNX27_PDZ measured with Jurkat or SH-SY5Y extracts. (F) Correlation between apparent affinities of SNX27_PDZ measured in two independent experiment series using Jurkat extracts. The second experiment series was measured on a different MS instrument using different number of replicates (n = 3). Direct proportionality was assumed between affinities, and the coefficient of proportionality (k), R² values, and PCC values are indicated in (E) and (F). Gray dashed line indicates the diagonal in (E) and (F). See fig. S3 and table S1 for additional data.