| Metadata |
| datasetIdentifier | PASS00704 |
| datasetType | MSMS |
| submitter | David Shteynberg <David.Shteynberg@systemsbiology.org> |
| submitter_organization | Institute for Systems Biology |
| lab_head_full_name | Robert L. Moritz |
| lab_head_email | Robert.Moritz@systemsbiology.org |
| lab_head_organization | Institute for Systems Biology |
| lab_head_country | United States |
| datasetTag | reSpect_ISB_Yeast |
| datasetTitle | reSpect Analysis of Saccharomyces cerevisiae Strain S288c ISB Lysate Sample |
| publicReleaseDate | 2015-06-09 00:00:00 |
| finalizedDate | |
| summary | This is the ISB generated data set of unfractionated yeast S288c lysate samples, used in the reSpect manuscript. |
| contributors | David Shteynberg, Luis Mendoza, Michael R. Hoopmann, Frank Schmidt, Zhi Sun, Eric W. Deutsch, and Robert L. Moritz |
| publication | Shteynberg D, Mendoza L, Hoopmann MR, Schmidt F, Sun Z, Deutsch EW, and Moritz RL, "reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra",JASMS, submitted
|
| growth | Yeast were grown under standard conditions, washed, lysed and then suspended in 100 mM ammonium bicarbonate buffer at a protein concentration of 0.5 mg/mL. |
| treatment | Yeast were grown under standard conditions. |
| extraction | The proteins were reduced with 5 mM dithiothreitol (DTT) for 30 minutes at 60oC, and alkylated with 15 mM iodoacetamide (IAM) for 30 minutes at room temperature in the dark. |
| separation | The peptides were separated by reverse phase liquid chromatography (RPLC) on an Easy-nLC 1000 (Thermo-Fisher Scientific, San Jose CA) prior to analysis using a Q-Exactive mass spectrometer (Thermo-Fisher Scientific). For each injection, 1 ug of peptides were loaded onto a 20 cm pulled fused-silica capillary column (75 um i.d.) packed with Reprosil-Pur C18 (3 um bead diameter, Dr. Maisch GmbH, Germany). |
| digestion | The proteins were digested to peptides for four hours at 37o C using trypsin (Promega sequencing grade, Madison, WI) at a ratio of 1:200. |
| acquisition | Peptides were eluted from the column using a binary mobile phase gradient, in which mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. The gradient was operated from 5 to 35% mobile phase B for 2 hours, followed by a 15 minute wash at 80% mobile phase B, and 30 minutes equilibration with 5% mobile phase B. The mass analyzer was operated with duty cycle consisting of a precursor scan from 300 to 1400 Th at 35,000 resolution followed by 20 HCD data-dependent acquisition (DDA) scans at 17,500 resolution. The DDA scans used an isolation window of 3.0 Th and a normalized collision energy of 30. Dynamic exclusion was set to 10 seconds and charge state exclusion of 1 and greater than 5 were used. |
| informatics | Dataset was searched with the Comet database search engine, using 25 ppm precursor tolerance with isotopic error enabled and using semi-tryptic enzymatic rules in the first pass search The search database utilized was UniProt yeast (2014-01) with an included set of randomized decoys. The search results were processed with PeptideProphet and iProphet versions bundled with TPP version 4.7.1. PeptideProphet was run with the ACCMASS model enabled, using NONPARAM option and specifying the DECOY=Random and DECOYPROBS decoy PSM handling options. All reSpect results, including the third and fourth round search results were processed along with the second-round search results so that there were sufficient data points for PeptideProphet and iProphet to model. The processing of reSpect results with PeptideProphet was done using the same options as with the first pass, but without the ACCMASS model. |
| instruments | Q-Exactive |
| species | Saccharomyces cerevisiae strain S288c |
| massModifications | C+57.02,M+15.9949 |
