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Analysis low level Hsp90 proteome by SILAC in yeast
Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic or non-genetic variation in populations. Despite information about Hsp90 protein-protein interactions, a global view of the Hsp90 regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the SILAC method coupled with mass spectrometry (MS) to quantify around 4000 proteins in low-Hsp90 cells and observed that 904 proteins were changed in their abundance by more than 1.5 fold. When compared with the transcriptome of the same population of cells, two-thirds of the mis-regulated proteins were observed to be affected post-transcriptionally, of which the majority were down-regulated. Further analyses indicated that the down-regulated proteins are highly conserved and assume central roles in cellular networks with a high number of interacting partners, suggesting that Hsp90 buffers genetic or non-genetic variation through regulating protein network hubs. The down-regulated proteins were enriched for essential proteins previously unknown to be Hsp90-dependent. Finally, we observed that down-regulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response respectively, providing a direct link between observed proteome regulation and cellular phenotypes.
Sample Processing Protocol
Normal and heavy SILAC-labeled yeast cells were mixed in a 1:1 ratio of cell numbers. The cells were resuspended in the lysis buffer (25 mM HEPES pH 7.5, 10 mM NaCl, 1 mM PMSF, and 1 mM DTT) and lysed using 0.5 mm glass beads with 5 beating cycles of 30 second-working and 30 second-cooling. To the mixture, 0.1% SDS was added and mixed by vortexing. All the above steps were performed at 4°C. The cell lysate was ultracentrifuged to remove the undissolved materials, and the protein was quantified using bicinchoninic acid (BCA) assay (BCA1, Sigma-Aldrich, St. Louis, MO). We aliquoted protein samples into 50 μg/each tube, and stored them at -80°C before MS analysis. Trypsin digestion and sample clean-up Samples containing 50 μg protein were dissolved in 20 μL of 50 mM NH4HCO3 containing 0.1% SDS. Each sample was reduced by 5 mM dithiothreitol (DTT) at 56°C for 30 min and then alkylated by 15 mM iodoacetamide at room temperature for 30 min. Excess iodoacetamide was quenched by additional 5 mM DTT at room temperature for 30 min. The solution was then brought to 200 μL with 10 mM NH4HCO3, and digestion was performed in the presence of 0.5 μg trypsin (Promega, Madison, WI) at 37°C overnight. The resulting peptide mixture was cleaned-up by a SOURCE 15RPC reverse phase (RP) micro-column (GE Healthcare, Uppsala, Sweden) and dried in a SpeedVac concentrator. Online 2-Dimensional Liquid Chromatography The comprehensive 2D-SCX-RP-LC system (Ultimate 3000, Thermo Fisher Scientific/Dionex, Germering, Germany) has been equipped with two gradient pumps (one for first and the other for second dimensional separation), one isocratic pump, one 10-port valve (installed with two RP-trapping columns) and one 6-port valve. The online two-dimensional peptide separation was achieved by operating both gradient pumps simultaneously and switching the 10-port valve every 65 min during the entire analysis. Briefly, protein digests dissolved in 50% acetonitrile containing 0.1% formic acid were loaded onto the SCX column (0.5 x 150 mm, pack with Luna-SCX particles from Phenomenex, Torrance, CA), which was operated at a flow rate of 1.5 μL/min. Peptides were eluted using a continuous concentration gradient of ammonium chloride in the presence of 0.1% formic acid and 30% acetonitrile. The salt gradient was segmented in 44 steps, 65 min for each, and eluted peptides from each step were then separated by the second dimensional reverse phase column (0.075 x 150 mm, pack with Synergi Hydro-RP particles from Phenomenex). The isocratic pump delivering 70 μL/min of solvent A (0.1% formic acid in water) was used for diluting the effluent of SCX column through a T-union and mixing tubing before it reached the trapping column (0.5 x 5 mm, packed with Symmetry300TM particles from Waters, Milford, MA). In the meantime, the other RP-trapping column was connected to the RP-analytical column and the effluent was analyzed by a mass spectrometer. Five minutes before each salt gradient step being completed, the gradient pump for the SCX separation stopped, and the six-port valve switched to allow the isocratic pump to wash away the residual salt solution in the RP-trapping column. The peptide-loaded trapping column was then switched to the RP analytical column to start a new step of separation, and the bound peptides were eluted at a flow rate of 300 nL/min with a complete acetonitrile gradient (elution, regeneration, and then re-equilibration) in the presence of 0.1% formic acid over 65 min. Mass spectrometry The effluent of the on-line 2D LC system was analyzed by an Orbitrap EliteTM Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a Nanospray Flex Ion Source. The instrument was operated in positive ion mode and the spray voltage was set to 1.8 kV. Full-scan MS spectra (m/z 400-m/z 2000) were acquired in the Orbitrap mass analyzer at a resolution of 60,000 at m/z 400, and the MS/MS spectra were acquired in the linear ion trap (LTQ). The m/z 445.1200, 462.1466, and 536.1654 cyclosiloxane peaks were used for lock mass calibration in the Orbitrap to improve mass accuracy. The values of automatic gain control (AGC) and the maximum accumulation times were 2e6 and 1000 ms for orbitrap, and 3000 and 120 ms for LTQ analyzer, respectively. Acquisition of MS/MS spectra was done in a data-dependent manner. Fifteen most intense ions in each full-scan MS spectrum with a minimal signal intensity of 5,000 were selected for collision induced fragmentation (CID) in LTQ with the following parameter settings: isolation width of 2.0 Da, normalized collision energy of 35%, activation Q of 0.25, and activation time of 10 ms. Each precursor ion was allowed to be sequenced once and then excluded dynamically for 40 seconds.
Data Processing Protocol
MS data Analysis The raw files were processed using the Proteome Discoverer software (version 1.3, Thermo Scientific, Waltham, MA). The CID spectra were searched against the UniProt database through Mascot search engine (version 2.2.2, Matrix Science Inc., Boston, MA). The search parameters were as follows: enzyme specificity was trypsin; a maximum of two miscleavages were allowed; precursor mass tolerance was 10 ppm; fragment mass tolerance was 0.6 Da; 13C6 L-Lys, 13C6 L-Arg, acetylation at protein N-terminus, glutamine to pyroglutamic acid conversion at peptide N-terminus, and methionine oxidation were set as variable modifications; and cysteine carbamidomethylation was set as a fixed modification. False discovery rate (FDR) of peptide/protein identifications were determined by employing a decoy database searching. Peptide identifications were then filtered with the requirements of rank 1 peptides with a minimum peptide length of seven amino acid residues. Only high confident peptide/protein identifications (FDR < 1%) were considered for further evaluation. Protein quantification was done with a SILAC workflow implemented in the Proteome Discover using the following parameters: limits of mass and retention differences between heavy and light pairs should be smaller than 4 ppm and 0.2 min, respectively; only unique peptides were used for quantification; and protein ratio was determined using the median of the corresponding peptide ratio. Finally, functional annotation of each identified protein was retrieved from the ProteinCenter server (Thermo Scientific). To investigate the proteomic effects caused by the dox treatment, we determined fold changes of SILAC signals upon dox treatment for all the identified proteins. First, the fold-changes were indicated as signal ratios of heavy-/light-labeled peptides (H/L). For one protein identity, we considered the median H/L ratio to represent the fold-change. Second, we normalized all of the fold changes with the median fold-change and log2-transformed the normalized fold change. Finally, we plotted the transformed data. To validate the reproducibility of the replicate SILAC experiment, we plotted fold-changes of the two experiments against each other to calculate the Pearson's correlation coefficient (Figure S2). We also considered the median SILAC signals (H/L for the 1st SILAC and L/H for the 2nd) of untreated/treated cells to represent the fold-change. We first log2-transformed the raw data of fold-changes and then plotted the transformed data.
Gopinath RK, You ST, Chien KY, Swamy KB, Yu JS, Schuyler SC, Leu JY. The Hsp90-dependent proteome is conserved and enriched for hub proteins with high levels of protein-protein connectivity. Genome Biol Evol. 2014 Oct 13. pii: evu226 PubMed: 25316598
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|1||38624||e.g. Control sample - Technical replicate #3||6521||238516||44120||509999||161993||