Figure 1. Schematics of SILAC-based experiments analyzed in our study.

(A) Small-molecule target identification workflow, as described in [30]. HeLa S3 cells were cultured in “heavy” medium, containing amino acids enriched in stable isotopes (¹³C and ¹⁵N), and “light” medium containing forms of natural isotope abundance. Both the heavy and light lysates were incubated with small-molecule loaded beads (referred to as the affinity matrix) while the light lysate contained the addition of a soluble form of the small-molecule that competed with the affinity matrix for binding to target proteins. The red ovals represent a target protein that was bound to all 3 small-molecule loaded beads shown in this schematic for the heavy lysate, but was competed off of 2 of the 3 beads in the light lysate. The green pentagons represent a protein that bound non-specifically to the beads in both the heavy and light lysates. Proteins bound to the affinity matrix were enriched by affinity pull-down, their relative abundances were quantified by LC-MS/MS, and targets were identified by analyzing the resulting abundance ratios, , using the empirical Bayes framework described in the text. (B) microRNA workflow, as described in [4]. HeLa cells were transfected with microRNAs or mock-transfected, and pulse-labeled after 8 hours with “medium” or “heavy” amino acid isotopes. After 24 hours, samples were combined and analyzed by LC-MS/MS. The red ovals represent a protein regulated by the microRNA that was depleted in the medium lysate, and the green pentagons represent an unregulated protein of equal abundance in both lysates. (C) Two affinity pull-down experiments performed at different soluble competitor concentrations of the protein kinase inhibitor k252a displayed distinct variances of log2 SILAC ratio distributions. Applying a commonly used threshold of 1.5-fold (log2 threshold of .58) to identify significant proteins inferred 66 non-kinases (indicative of false-positives) for the high-variance experiment compared to 11 for the low-variance experiment, suggesting the necessity of experiment-specific models. (inset) The red curve represents the same k252a experiment, performed at .25x concentration, as displayed in the main plot, and the grey curve represents a replicate performed on the same cellular lysate with the same k252a concentration. This replicate experiment displayed a shift in the overall distribution due to non 1-to-1 mixing of heavy and light samples, which should be accounted for by appropriate normalization.