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PeptideAtlas provides a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments collected for human, mouse, yeast, and several other organisms.
(last updated: Nov 28, 2017)
Data or Model Molecule| Accession | Sample Title | Summary | Organism | Characteristics | Instrument | Treatment | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| PAe000084 | Saccharomyces cerevisiae (yeast) treated with mating hormone, ICAT | Catalogue changes in budding yeast (S. cerevisiae) protein expression following brief treatment with mating hormone, with the endpoint of identifying translationally controlled proteins. baseline (light ICAT label) is protein from an asynchrnous culture same harvest and chromatographic methods as with TSAA timepoint experiments, but in this case measurements taken 30 minutes after alpha-factor application (no alpha-factor removal); baseline (light ICAT label) is protein from an asynchrnous culture | Yeast | BY1782 (MATa cdc15-2 tyr1 leu2 ura3 his7 gal1), BY2125 (MATa ade2-1 his3-11 leu2-3, 112 trp1-1 ura3 can1-100 ssdl-d) | LTQ | Cultures were grown at 25°C in a shaking incubator except during the cdc15 arrest (37°C) and growth of cells for the β-galactosidase assays (30°C). cdc15 Synchronization -- Cells were grown in 400 ml of YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) to a density of 5–8×10⁶cells/ml, and then the cultures were shifted to 37°C for 3 h. To release cells from arrest, flasks were swirled rapidly in an ice-water bath so that the culture temperature reached 25°C in less than 1 min. Two separate experiments were conducted: one in which samples for probe preparation were taken every 10 min from 0 to 200min and one in which samples were taken every 20 min from 0 to 60min. The collection of samples took place over several months in four sets (0–50 min, 60–100 min, 110–150 min, and 160–200 min). At the same time that samples for TSAA were taken, aliquots of cells were also removed for bud counts and total RNA isolation. α-Factor Synchronization—Cells were grown to a density of 5–8×10⁶cells/ml at which point α-factor was added. The cells were fully arrested after 2 h. To release cells from arrest they were centrifuged for 2 min at 3000×g and resuspended in 400 ml of fresh medium. Two separate experiments were conducted: one in which samples were taken every 10 min for 0 to 190 min and one in which samples were taken every 10 min from 0 to 50 min. For each time point, RNA isolated from three separate cultures was pooled before labeling. At the same time that samples for TSAA were taken, aliquots of cells were removed for bud counts and total RNA isolation. | Trypsin | All steps took place in a 4°C cold room. To prepare polysomes, ~100 ml of culture was poured into a flask containing ~75 ml of crushed frozen YEPD. The flask was swirled rapidly on ice for 1–2 min and then centrifuged for 2 min at 3000 × g to pellet the cells. The pellet was resuspended in 10 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 100mM NaCl, 30mM MgCl₂, ] 500µg/ml heparin, 50µg/ml cycloheximide) and centrifuged a second time. The pellet was resuspended in 1 ml of lysis buffer and transferred to a 15-ml conical tube containing 2 g of glass beads (~500-µm diameter, Sigma). The tubes were vortexed eight times for 30 s each. Tubes were placed on ice for 30 s between rounds of vortexing. Tubes were centrifuged for 1 min in a clinical centrifuge to pellet the beads, and the supernatant was removed. Lysis buffer (0.5 ml) was added to the beads, the beads were vortexed for 15 s, and the tube was centrifuged again to pellet the beads. The supernatant was removed and added to the first supernatant. The pooled supernatants were centrifuged in a microcentrifuge at top speed (20,000 × g) for 1 min to pellet debris, and the supernatant was transferred to a fresh tube. The A₂₆₀ of the supernatant was measured. For the cdc15 experiments 50–60 absorbance units were loaded per gradient. For the α-factor experiments, 25 absorbance units were loaded per gradient. | The samples were loaded on a 10-ml linear gradient of 7–47% sucrose with a 1-ml 2 M sucrose cushion at the bottom and centrifuged for 2 h in an SW40 Ti swinging bucket rotor at 4°C and 39,000 rpm. The gradient buffer consisted of 50 mM Tris acetate, pH 7.0, 50 mM NH₄Cl, 12 mM MgCl₂, and 1 mM dithiothreitol. After centrifugation, the gradients were collected into 24 0.5-ml fractions. Fifty microliters of 3 M NaOAc, pH 5.2, and 50 µl of 10% SDS were added to each fraction. During fractionation a running trace of A₂₅₄ was recorded to monitor the polysome profile. | PMID:12684541 |
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