Fig. 3.
SILAC-BioID: Enrichment quantified against controls. A, Supplemented biotin was used by the Asc1-BirA* fusion protein to covalently biotinylate proteins in reach (up to ∼35 Å). After cell lysis in the presence of 4% SDS biotinylated proteins were enriched via biotin affinity capture and afterwards digested with trypsin. The resulting peptides were subjected to LC-MS analysis for protein identification and for the determination of biotinylated lysine residues. SILAC quantification was performed with the MaxQuant software (39). Additional database search with the Proteome Discoverer software enhanced the identification of biotinylated sites. To improve the coverage of biotinylated peptides, half of the peptide solution was subjected to additional biotin affinity capture and LC-MS analysis. Peptide samples without biotin affinity capture were also analyzed by LC-MS. The resulting proteome values were used to normalize affinity capture SILAC ratios against total protein abundances. Modified from Smolinski and Valerius (74). B, All four BioID-experiments were performed with triple SILAC to quantitatively compare cells of three different cultures in one batch. The birA* and wt-ASC1 strains served as negative controls. Alterations in the Asc1p-neighborhood on glucose starvation and heat stress (37 °C) were determined against ASC1-birA* at exponential growth (2% glucose, 30 °C) as reference and birA* at stress as negative control. The neighborhood of Asc1DE-BirA*p was compared with ASC1-birA* as reference and birA* as negative control. Yeast strains were individually cultivated in the presence of 10 μm biotin and were labeled with lysine and arginine isotope variants as indicated. All genes were expressed from high copy number plasmids in a Δarg4 Δlys1 strain background to ensure exclusive incorporation of the labeled amino acids into all proteins. The number of biological replicates is indicated for each experiment. °One replicate of the “heat stress” BioID experiment was performed with a label swap: ASC1-birA*, 37 °C (heavy), ASC1-birA*, 30 °C (medium), birA*, 37 °C (light).