Fig. 2. Single-point nHU for rapid apparent affinity measurements.

(A) Demonstration of single-point nHU-WB using 12 different biotinylated PBM peptides (baits) or biotin (control) saturated streptavidin resin and total Jurkat extracts. Supernatant fractions were probed with specific antibodies against endogenous full-length PDZ domain–containing proteins SCRIB and SNX27. (B) Results of the nHU-WB experiment presented in (A). Correlation between in vitro fragmentomic affinities measured using PBM peptides and isolated PDZ domains (12)⁠ and apparent affinities measured with nHU between PBM peptides and full-length proteins. In the case of SCRIB (right), the site-specific fragmentomic affinities were combined assuming simple additivity. Direct proportionality was assumed between affinities (gray dashed line), and the coefficient of proportionality (k), coefficient of determination (R²) values, and Pearson correlation coefficient (PCC) values are indicated. Note the negative R² value in the case of SCRIB, which indicates that a better fit could be obtained with a model with nonzero intercept; however, the physical basis of such model would be difficult to justify. Affinities were determined on the basis of three replicates. See fig. S1 and table S1 for additional data.