PRIDE Assigned Tags:Biological Dataset
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Quantitative proteomic analyses of budding yeast cells harboring an additional chromosome
Phenotypic variability is a hallmark of diseases involving chromosome gains and losses, such as Down Syndrome and cancer. Allelic variances have been thought to be the sole cause of this heterogeneity. Here, we systematically examine the consequences of gaining and losing single or multiple chromosomes to show that the aneuploid state causes non-genetic phenotypic variability. Yeast cell populations harboring the same defined aneuploidy exhibit heterogeneity in cell cycle progression and response to environmental perturbations, which we show to be partly due to gene copy number imbalances. Thus, subtle changes in gene expression severely impact the robustness of biological networks and cause alternate behaviors when they occur at a large scale. Because trisomic mice also exhibit variable phenotypes, we further propose that non-genetic individuality is a universal characteristic of the aneuploid state that could contribute to variability in presentation and treatment responses of diseases caused by aneuploidy.
Sample Processing Protocol
Proteins were reduced with 10mM dithiothreitol (Sigma) for 1h at 56oC and then alkylated with 55mM iodoacetamide (Sigma) for 1h at 25oC in the dark. Proteins were then digested with modified trypsin (Promega) at an enzyme/substrate ratio of 1:50 in 100mM ammonium acetate, pH 8.9 at 25oC overnight. Trypsin activity was halted by addition of acetic acid (99.9%, Sigma) to a final concentration of 5%. After desalting using a C18 Sep-Pak Plus cartridge (Waters), peptides were lyophilized in 400ug aliquots and stored at -80oC. Peptide labeling with TMT 6plex (Thermo) was performed per manufacturer’s instructions. Lyophilized samples were dissolved in 70 μL ethanol and 30 μL of 500 mM triethylammonium bicarbonate, pH 8.5, and the TMT reagent was dissolved in 30 μL of anhydrous acetonitrile. The solution containing peptides and TMT reagent was vortexed and incubated at room temperature for 1 h. Samples labeled with the ten different isotopic TMT reagents were combined and concentrated to completion in a vacuum centrifuge. The TMT-labeled peptide pellet was fractioned via high-pH reverse phase HPLC. Peptides were resuspended in 100uL buffer A (10mM TEAB, pH8) and separated on a 4.6mm x 250 mm 300Extend-C18, 5um column (Agilent) using an 90 minute gradient with buffer B (90% MeCN, 10mM TEAB, pH8) at a flow rate of 1ml/min. The gradient was as follows: 1-5% B (0-10min), 5-35% B (10-70min), 35-70% B (70-80min), 70% B (80-90min). Fractions were collected over 75 minutes at 1 minute intervals from 10 min to 85 min. The fractions were concatenated into 15 fractions non-contiguously (1+16+31+46+61, 2+17+32+47+62, etc). The fractions were speed-vac (Thermo Scientific Savant) to near dryness.
Data Processing Protocol
Peptides were loaded on a precolumn and separated by reverse phase HPLC using an EASY- nLC1000 (Thermo) over a 140 minute gradient before nanoelectrospray using a QExactive mass spectrometer (Thermo). The mass spectrometer was operated in a data-dependent mode. The parameters for the full scan MS were: resolution of 70,000 across 350-2000 m/z, AGC 3e6, and maximum IT 50 ms. The full MS scan was followed by MS/MS for the top 10 precursor ions in each cycle with a NCE of 34 and dynamic exclusion of 30 s. Raw mass spectral data files (.raw) were searched using Proteome Discoverer (Thermo) and Mascot version 2.4.1 (Matrix Science). Mascot search parameters were: 10 ppm mass tolerance for precursor ions; 15 mmu for fragment ion mass tolerance; 2 missed cleavages of trypsin; fixed modification were carbamidomethylation of cysteine and TMT 10plex modification of lysines and peptide N-termini; variable modification was methionine oxidation. TMT quantification was obtained using Proteome Discoverer and isotopically corrected per manufacturer’s instructions, and were normalized to the mean of each TMT channel. Only peptides with a Mascot score greater than or equal to 25 and an isolation interference less than or equal to 30 were included in the data analysis.
Beach RR, Ricci-Tam C, Brennan CM, Moomau CA, Hsu PH, Hua B, Silberman RE, Springer M, Amon A. Aneuploidy Causes Non-genetic Individuality. Cell. 2017 Apr 6;169(2):229-242.e21 PubMed: 28388408
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