| Metadata |
| datasetIdentifier | PASS00504 |
| datasetType | SRM |
| submitter | Lucia Espona Pernas <espona@imsb.biol.ethz.ch> |
| submitter_organization | IMSB ETH Zürich |
| lab_head_full_name | Ruedi Aebersold |
| lab_head_email | aebersold@imbs.biol.ethz.ch |
| lab_head_organization | IMSB ETH Zürich |
| lab_head_country | Switzerland |
| datasetTag | NSELEV_YEAST_PROTEOM |
| datasetTitle | SWATH Analysis of Yeast Proteome over Time in Response to Osmotic Stress: SRM Dataset |
| publicReleaseDate | 2015-01-05 00:00:00 |
| finalizedDate | 2015-01-14 08:36:20 |
| summary | We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection DIA datasets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. To validate the protein fold changes obtained by SWATH-MS, we quantified a subset of 100 proteins in the 18 osmotic shock time course samples using SRM. |
| contributors | Nathalie Selevsek, Ching-Yun Chang, Ludovic C Gillet, Pedro Navarro, Oliver M Bernhardt, Lukas Reiter, Olga Vitek, Ruedi Aebersold |
| publication | Mol Cell Proteomics. 2015 Jan 5. pii: mcp.M113.035550. [Epub ahead of print]
Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-MS.
PubMedID: 25561506
|
| growth | Three series of six cultures each from the yeast strain BY4741 MATa his3Δ leu2Δ met15Δ ura3Δ were grown in SD medium until they reached an A600 of 0.8. To apply the osmotic shock, 0.4 M NaCl was added to each 50 ml culture and after 0 min (T0), 15 min (T1), 30 min (T2), 60 min (T3), 90 (T4) and 120 min (T5) the cells were harvested. Indeed, the culture media was quenched by addition of trichloroacetic acid (TCA) to a final concentration of 6.25 % and the cells were harvested by centrifugation at 1500 g for 5 min at 4°C. The supernatants were discarded and the cell pellets were washed three times by centrifugation with cold (-20°C) acetone to remove interfering compounds. The final cell pellets were resolubilized in lysis buffer containing 8 M urea, 0.1 M NH4HCO3 and 5 mM EDTA and cells were disrupted by glass bead beating (5 times 5 minutes at 4°C). |
| treatment | To apply the osmotic shock, 0.4 M NaCl was added to each 50 ml culture and after 0 min (T0), 15 min (T1), 30 min (T2), 60 min (T3), 90 (T4) and 120 min (T5) the cells were harvested. Indeed, the culture media was quenched by addition of trichloroacetic acid (TCA) to a final concentration of 6.25 % and the cells were harvested by centrifugation at 1500 g for 5 min at 4°C. The supernatants were discarded and the cell pellets were washed three times by centrifugation with cold (-20°C) acetone to remove interfering compounds. The final cell pellets were resolubilized in lysis buffer containing 8 M urea, 0.1 M NH4HCO3 and 5 mM EDTA and cells were disrupted by glass bead beating (5 times 5 minutes at 4°C). |
| extraction | The supernatants were discarded and the cell pellets were washed three times by centrifugation with cold (-20°C) acetone to remove interfering compounds. The final cell pellets were resolubilized in lysis buffer containing 8 M urea, 0.1 M NH4HCO3 and 5 mM EDTA and cells were disrupted by glass bead beating (5 times 5 minutes at 4°C). The total protein amount from the pooled supernatants was estimated by BCA Protein Assay Kit (Thermo, Rockford, US). Yeast proteins were reduced with 12 mM dithiotreitol at 37°C for 30 min and alkylated with 40 mM iodoacetamide at room temperature in the dark for 30 min. |
| separation | |
| digestion | Samples were diluted with 0.1 M NH4HCO3 to a final concentration of 1.5 M urea and the proteins were digested with sequencing grade porcine trypsin (Promega) at a final enzyme:substrate ratio of 1:100. Digestion was stopped by adding formic acid to a final concentration of 1%. Peptide mixtures were desalted using reverse phase cartridges Sep-Pak tC18 (Waters, Milford, MA) according to the following procedure; wet cartridge with 1 volume of 100% methanol, wash with 1 volume of 80% acetonitrile, equilibrate with 4 volumes of 0.1% formic acid, load acidified digest, wash 6 volumes of 0.1% formic acid, and elute with 1 volume of 50% acetonitrile in 0.1% formic acid. Peptides were dried using a vacuum centrifuge and resolubilized in 100 µl of 0.1% formic acid and frozen at -20°C. For in depth-fractionation experiments of NaCl-untreated yeast cells, the peptide mixtures were separated by off gel electrophoresis (OGE) using a pH 3–7 IPG strip (Amersham Biosciences) and a 3100 OFFGEL Fractionator (Agilent Technologies) with collection in 12 wells and then submitted for C18 clean-up. All samples were spiked with the retention time standard peptides iRT-Kit (Biognosys, Schlieren, Switzerland). |
| acquisition | |
| informatics | To validate the protein fold changes obtained by SWATH-MS, we quantified a subset of 100 proteins in the 18 osmotic shock time course samples using a another targeted proteomics approach namely SRM. We selected 50 proteins that were identified by SWATH-MS as up-regulated (n= 25) and down-regulated (n= 25), respectively, and distributed among the enriched pathways. For normalization purpose, we also added 50 proteins that were not found regulated across the time course study. For each protein, we choose the optimal peptides confidently identified in SWATH-MS, together with their corresponding fragment ions and quantified them by SRM across the 18 yeast samples. We then integrated the fragment ion traces of all peptides detected with SRM and re-extracted the peak area of the same peptide measured by SWATH-MS. For consistency in the data analysis we used the skyline software, since it allowed us to process both data sets with the same integration/quantification parameters. We then determined the fold changes between 15 min (T1), 30 min (T2), 60 min (T3), 90 min (T4), 120 min (T5) and the initial time point 0 min (T0) and upon addition of salt by SWATH-MS and SRM. Figure 6A shows log2 fold change profiles of representative examples for up-regulated and down-regulated proteins by SRM and SWATH-MS. To further pinpoint the proteins that were significantly changing in abundance between the different time points, we applied SRMstats, a statistical modeling framework for protein significance analysis previously designed for SRM experiments. |
| instruments | TSQ Quantum |
| species | Saccharomyces cerevisiae (Baker's yeast) |
| massModifications | static: C+57.021464
variable: M-63.9983 |
