Fig. 1.

Identifying specific SM-protein interactions with quantitative proteomics. (A) SILAC identifies specific protein interactions with SM baits. Cell populations are fully labeled with light (black) and heavy amino acids (red) and lysates incubated either with SM-loaded beads (SM-Beads) and soluble SM competitor or SM-Beads alone. Proteins interacting directly with the SM or via secondary and/or higher order interactions (marked “S” for specific) will be enriched in the heavy state over the light and will be identified with differential ratios. Nonspecific (NS) interactions of proteins will be enriched equally in both states and have ratios close to 1. (B) Experimental mass spectra showing specific protein interactions with the immunophilin ligand, AP1497. (Left) A peptide from FKBP4, a known binding partner to FK506, is observed with a highly differential ratio. (Right) In contrast, a histone H1.3 peptide is identified with a ratio close to 1, indicating no specific binding to the soluble SM competitor.