Pull-down of Exo1-TAP from yeast meiotic cells and analysis of phosphorylated residues
UID: 10614
- Description
- Description from the ProteomeXchange: "Exo1 tagged with the TAP sequence was pulled down from synchronous ndt80∆ meiotic cells after induction of the CDC5 kinase at t = 8 h 15 in meiosis (1 h 15 after CDC5 induction), in order to identify its phosphorylated residues"
PRIDE Sample Processing Protocol: "2.1010 cells of VBD2070 strain were harvested after 8 h 15 min in sporulation medium and processed for TAP pull-down in the presence of 125 U/mL benzonase. 1500 μL of PanMouse IgG magnetic beads (Thermo Scientific) were washed 1:1 with lysis buffer, preincubated in 100 μg/ml BSA in lysis buffer for 2 h at 4°C and then washed twice with 1:1 lysis buffer. The lysate was cleared by centrifugation at 8000 g for 10 min at 4°C and incubated overnight at 4°C with washed PanMouse IgG magnetic beads. The magnetic beads were washed four times with 8 mL of wash buffer (20 mM HEPES/KOH pH7.5; 150 mM NaCl; 0.5% Triton X-100; 5% Glycerol; 1 mM MgCl2; 2 mM EDTA; 1 mM PMSF; 1X Complete Mini EDTA-Free (Roche)). Beads were then resuspended in 40 μl of Laemmli buffer and boiled for 10 min at 75°C. Proteins were separated by SDS-PAGE, stained with colloidal blue, and the band predicted to contain Exo1-TAP was excised and processed. Excised gel slice was washed and proteins were reduced with 10 mM DTT prior to alkylation with 55 mM iodoacetamide. After washing and shrinking of the gel pieces with 100% acetonitrile, in-gel digestion was performed using trypsin/LysC (0.1µg) overnight in 25 mM ammonium bicarbonate at 30°C. Peptide are then extracted using 60/35/5 MeCN/H2O/HCOOH, vacuum concentrated to dryness and reconstituted in loading buffer A (2/98 MeCN/H2O + 0.05% TFA) prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The extracted peptides were chromatographically separated using an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Scientific). Peptides were loaded onto a C18-reversed phase column (75-μm inner diameter × 2 cm; nanoViper Acclaim PepMapTM 100, Thermo Scientific) with buffer A’, separated and MS data acquired using Xcalibur software. Peptides separation was performed over a linear gradient of 91 min from 2% to 30% buffer B (75-μm inner diameter × 50 cm; nanoViper Acclaim PepMapTM RSLC, 2 μm, 100Å, Thermo Scientific) at a flow rate of 300 nL/min. Full-scan MS was acquired in the Orbitrap analyzer with a resolution set to 120,000 and the top20 intense ions were subjected to Orbitrap for futher fragmentation via high energy collision dissociation (HCD) activation and a dynamic exclusion on 20 s."
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Dataset files accessible through FTP [file transfer protocol] link.
Accession #: PXD014185Access via PRIDEDataset files accessible through FTP link.
Accession #: PXD014185 - Access Restrictions
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Free to All
- Access Instructions
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- Data Type
- Equipment Used
- Software Used
- Dataset Format(s)
- DAT, RAW, MGF, MSF
- Dataset Size
- 61 MB (DAT), 66 MB (MGF), 180 MB (MSF), 626MB (RAW)
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