), and ubiquitylation sites (supplemental Table S5). Information Analysis–Statistical significance was calculated working with the R environment. Gene Ontology (GO) term association and enrichment evaluation have been performed employing the Database for Annotation, Visualization and Integrated Discovery (DAVID) (39). Phosphorylation and di-Gly-modified sites had been clustered based on their dynamic behavior making use of GProx (40). Amino acid motif enrichment within clusters was analyzed making use of IceLogo (41). To construct a protein?protein interaction network, the STRING database method was used (42). Functional protein interaction networks had been visualized using Cytoscape (43).RESULTSExperimental Strategy–In this study we analyzed rapamycin-induced changes in protein, ubiquitylation, and phosphorylation abundance at two time points (1 h and 3 h) in the model organism S. cerevisiae (Fig. 1A). Proteome adjustments were quantified in an unbiased (non-hypothesis-driven) manner working with a SILAC-based proteomic strategy (44). Protein extracts from “light” (handle, mock treated), “medium” (1 h, 200 nM rapamycin), and “heavy” (3 h, 200 nM rapamycin) SILAC-labeled yeast samples were combined in equal amounts and digested to peptides using Lys-C and trypsin. Di-Gly-modified peptides were enriched using a monoclonal antibody directed toward the di-Gly remnant (16, 17, 21). Phosphorylated peptides had been enriched employing TiO2-based metal affinity chromatography (32, 33). So as to cut down sample complexity, peptides have been fractionated employing microtip SCX columns (28, 45).4-Bromo-3,5-dimethylphenylboronic acid Price Peptides had been analyzed by suggests of high-pressure nano-flow reversed phase chromatography straight connected to a quadrupole-Orbitrap mass spectrometer (Q Exactive) (34, 35).Gaboxadol (hydrochloride) Order Computational analysis of MS information was performed working with MaxQuant (36, 37), enabling a maximum false discovery price of 1 .PMID:24563649 We utilized stricter criteria for PTM evaluation by requiring a minimum posterior error probability score of 0.01 and localization probability of 0.75 for phosphorylated peptides or 0.9 for di-Gly-modified peptides. From three biological replicates, we quantified 3590 proteins, 2299 di-Gly modification internet sites, and 8961 phosphorylation web sites (supplemental Table S1). The Rapamycin-regulated Proteome–In order to provide an in-depth proteomic evaluation of rapamycin-treated yeast cells, we sought to quantify changes in protein abundance.Molecular Cellular Proteomics 13.Phosphorylation and Ubiquitylation Dynamics in TOR SignalingALight No Rapamycin Medium 1h Rapamycin Proteins mixed 1:1:1 Heavy 3h RapamycinBProteomen = 3590 230 2578 171 119 Experiment 3 n = 2932 64 95 Experiment two n =Experiment 1 n = 3169Lys-C digestionPhosphoproteomen = 8961 Experiment 1 n = 5931 333 515 4275 Trypsin digestion 808 1882 Experiment 3 n = 7783 818 330 Experiment 2 n =ProteomePhosphoproteomeUbiquitylomephosphopeptide enrichment (TiO2)Di-glycine peptides immunoenrichment SCX fractionationDi-Gly proteome (Ubiquitylome)SCX fractionation SCX fractionation n = 2299 Experiment 1 n = 1499 458 104 129 LC-MS/MS Information evaluation Experiment 3 n = 904 394 543 128 543 Experiment two n =FIG. 1. Proteome, phosphoproteome, and ubiquitylome evaluation of rapamycin-treated yeast. A, experimental outline. Exponentially growing yeast cells had been metabolically labeled with lysine0 (light), lysine4 (medium), or lysine8 (heavy). Rapamycin was added to 0.two mM, and cells have been harvested in the indicated time points. Equal amounts of proteins were mixed and digested beneath denaturing circumstances applying e.