| Metadata |
| datasetIdentifier | PASS01559 |
| datasetType | SWATH |
| submitter | Ulrike Kusebauch <ukusebauch@systemsbiology.org> |
| submitter_organization | Institute f. Systems Biology |
| lab_head_full_name | Robert Moritz |
| lab_head_email | Robert.Moritz@systemsbiology.org |
| lab_head_organization | Institute f. Systems Biology |
| lab_head_country | United States |
| datasetTag | HSalinarum |
| datasetTitle | Analysis of Halobacterium salinarum by SWATH-MS |
| publicReleaseDate | 2020-07-09 00:00:00 |
| finalizedDate | 2020-07-09 08:25:00 |
| summary | SWATH-MS analysis of Halobacterium salinarum sp. NRC-1 sampled at 4 time points (see Growth Protocol). Acquired data include three biological replicates for each time point and three technical replicates. |
| contributors | Adrián López García de Lomana, Ulrike Kusebauch, Arjun V. Raman, Min Pan, Serdar Turkarslan, Alan P.R. Lorenzetti, Robert L. Moritz and Nitin S. Baliga |
| publication | Selective translation of low-abundance and upregulated transcripts in prokaryotes, in preparation |
| growth | Wild-type Halobacterium salinarum sp. NRC-1 was cultured in liquid nutrient-rich complex media (CM): 250 g/L NaCl, 20 g/L MgSO4•7H2O, 3 g/L sodium citrate, 2 g/L KCl and 10 g/L peptone (Oxoid, United Kingdom) made with distilled water. Cultures were inoculated to a starting optical density at 600 nm (OD600) of 0.02 with starter culture of OD600 0.5 which was derived from a single colony. Cultures were grown in unbaffled flasks in which 40% of the flask volume is occupied by the culture. Cultures were grown at 37°C, shaken at 220 rpm, and illuminated at ~20 μmol/m2/sec in Innova9400 incubators (New Brunswick). Triplicate cultures were grown, and samples were harvested at four time points. The four time points were selected to represent the early exponential phase (OD600 0.2; 14.3 hours), mid-exponential growth (OD600 0.5; 21.5 hours), late exponential phase (OD600 0.8; 28.8 hours), and stationary phase (40.8 hours). The final time point was selected to be 12 hours past the late exponential phase, since OD600 readings are not representative of cell growth in H. salinarum in stationary phase [24]. At each time point, whole cells were collected by centrifugation by RNA-sequencing, ribosome profiling and mass spectrometry (MS) proteomics. |
| treatment | |
| extraction | Cells were collected by centrifugation (8,000 x g, 2 min, 4 °C), cell pellets were resuspended in 3.4 M KCl, 100 mM MgCl2, 10 mM Tris-HCl pH 7.4 and titrated, followed by sonication and centrifugation (14,000 x g, 10 min, 4 °C). Supernatants were collected and treated with RQ1 DNase (Promega), followed by centrifugation (14,000 x g, 10 min, 4 °C). Ribosome-bound RNA was generated by treating the lysate with RNAse I and quenching the reaction with Superase-In RNase Inhibitor. Macromolecular complexes were collected by spin column isolation (MicroSpinTM S-400 HR, GE), elution was performed by centrifugation (600 x g, 2 min, room temperature), samples snap-frozen in liquid nitrogen and stored at -80°C. Elution was split into two aliquots, one for ribosome footprints sequencing and one for proteome analysis |
| separation | |
| digestion | The protein content of the samples was determined by bicinchoninic acid assay (Thermo-Fisher). Proteins were reduced (5 mM Dithiothreitol, 45 min, 37°C), alkylated (14 mM iodoacetamide, 30 min, room temperature, darkness) and digested with trypsin (1:50 enzyme:substrate ratio, 37°C, 16 h). Samples were desalted with tC18 SepPak cartridges (Waters). |
| acquisition | Samples were analyzed with a TripleTOF® 5600+ system equipped with a Nanospray-III® Source (Sciex) and an Eksigent Ekspert™ nanoLC 425 with cHiPLC® system in trap-elute mode (Sciex). Peptides were separated with a gradient from 3% to 33% 0.1% formic acid in acetonitrile (v/v) in 120 min. Data were collected in MS/MSALL SWATH™ Acquisition mode using 100 variable acquisition windows. |
| informatics | |
| instruments | Sciex TripleTOF 5600 |
| species | Halobacterium Salinarum NRC-1 |
| massModifications | static: C+57.021464, variable: M++15.9949 |
