Rapamycin + Lysis Trypsinize 16 LC-MS/MS Down-regulated “Light” Labelling “Heavy” 4 biological replicates A. Soulard et al. A - Lysis preparative SDS-PAGE 1 . . . 16 IMAC 1 C 2 8 . 0 2 5 6 . 0 1 B 7 8 . 0 1 5 2 . 5 8 6 . 5 MS/MS scans 284149 unique quantified phospho-sites 2201 pS 370 pT 36 pY Up-regulated Figure 1. Quantitative analysis of the rapa- mycin-sensitive phosphoproteome by SILAC. (A) Schematic overview of the experimental approach for the identiﬁcation of TORC1 reg- ulated phosphorylation sites in S. cerevisiae. Two yeast cultures (strain YPJ2) were metabol- ically labeled with 12C -14N2-lysine/12C6-argi- 6 nine (light) or 13C -15N2-lysine/13C6-arginine 6 (heavy). The heavy culture was treated for 15 min with rapamycin. Extracts were mixed in a 1:1 ratio and separated by preparative SDS- PAGE. The gel was horizontally sliced into 16 sections and in-gel–digested, and phosphopep- tides were IMAC-enriched and analyzed in an LTQ-Orbitrap. (B) Merged data from the four experiments revealed 972 phosphoproteins, corresponding to 2487 unique phosphopeptides and 2607 unique phosphosites. (C) Analysis by Motif-X of all regulated phosphopeptides. stood. In particular, very few substrates of either TORC1 or its direct effectors such as the AGC kinase SCH9 are known. To identify proteins whose phosphorylation changes in a TORC1-dependent manner, we performed unbiased quanti- tative phosphoproteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC; Ong et al., 2002). Extracts from rapamycin-treated untreated yeast cells were separated by preparative SDS gel electrophoresis, phos- phopeptides were enriched by IMAC, and the resulting phosphopeptide pools were analyzed by LC-MS/MS (Fig- ure 1A). Unique phosphopeptides (n 2487) and unique phosphorylation sites (n 2607), corresponding to 972 phosphoproteins, were identiﬁed and quantiﬁed (Figure 1B and Supplementary Table 3). Our phosphoproteome com- pares favorably to previously published yeast phosphopro- teomes (Gruhler et al., 2005; Li et al., 2007; Wilson-Grady et al., 2008) and includes both high- and low-abundance phos- phoproteins (Supplementary Figure 1B; Ghaemmaghami et al., 2003). Of the 972 phosphoproteins identiﬁed (Supple- mentary Table 3), 78 were signiﬁcantly hyperphosphory- lated, and 55 were hypophosphorylated upon rapamycin 3478 treatment (Supplementary Tables 4 and 5, respectively). To accurately quantify the changes in phosphorylation, the fold change in phosphorylation was systematically compared with the fold change in expression for each protein (Supple- mentary Tables 6 – 8). Analysis of the data revealed that many rapamycin-regulated phosphoproteins had been already genetically or biochemically linked to TORC1 signaling (Sup- plementary Figure 1, C and D), providing further validation of our phosphoproteome. During the preparation of this manu- script, another study describing the rapamycin-regulated phos- phoproteome in yeast was published (Huber et al., 2009). A comparison of the two studies revealed surprisingly little over- lap. Although the total number of rapamycin-regulated pro- teins was very similar in the two studies (133 vs. 129), only 29 proteins were common to both studies (Supplementary Figure 1, C and D). Thus, the two studies provide different but com- plementary rapamycin-regulated phosphoproteomes. To determine if TORC1 controls phosphorylation of speciﬁc motifs, we analyzed the sequences surrounding the rapamycin-regulated phosphosites, with the Motif-X algorithm (Schwartz and Gygi, 2005). For the peptides Molecular Biology of the Cell