PRIDE Assigned Tags:Biological Dataset
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Central bearded dragon hibernation label-free LC-MS/MS
Hibernation is a complex physiological state often exploited by animals under adverse environmental conditions. It involves large changes in metabolism and cellular function, with many stress responses modulated to tolerate physiological challenges that would otherwise be lethal. This study explores transcriptomic and proteomic profiling of hibernation in a reptile (the central bearded dragon; Pogona vitticeps) throughout the hibernation season. To better understand molecular dynamics of this remarkable process, total RNA and protein was analyzed in brain, heart and skeletal muscle at three time points; late hibernation, two days post-arousal, and two months post-arousal.
Sample Processing Protocol
Total protein was extracted from 50 mg of tissue. Tissue extracts were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA), cOmplete® and EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland) using T10 Basic ULTRA-TURRAX® Homogenizer (IKA, Staufen im Breisgau, Germany). Protein concentrations were determined using a Qubit 2.0 Fluorometer (Thermofisher, Waltham, Massachusetts, USA).
Data Processing Protocol
Protein extracts were analyzed at the Bioanalytical Mass Spectrometry Facility at the Mark Wainwright Analytical Centre (UNSW, Australia) using label-free quantification mass spectrometry using standard protocol. Briefly, samples were digested with Trypsin (MS Grade, Thermofisher) and separated by nanoLC using an Ultimate nanoRSLC UPLC and autosampler system (Dionex, Amsterdam, Netherlands). Samples (2.5 µl) were concentrated and desalted with a micro C18 precolumn with H2O:CH3CN (98:2, 0.1 % TFA) at 15 µl/min and a fritless nano column (75 µm x 15cm) containing C18-AQ media (Dr Maisch, Ammerbuch-Entringen Germany). Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1 % formic acid) to H2O:CH3CN (64:36, 0.1 % formic acid) at 200 nl/min over 60 min. 2000 volts was applied to low volume titanium union and the tip positioned ~ 0.5 cm from the heated capillary (T=275°C) of an Orbitrap Fusion Lumos (Thermo Electron, Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Fusion Lumos operated in data dependent acquisition mode (DDA). A survey scan m/z 350-1750 was acquired in the orbitrap (resolution = 120,000 at m/z 200, with an accumulation target value of 400,000 ions) and lockmass enabled (m/z 445.12003). Data-dependent tandem MS analysis was performed using a top-speed approach (cycle time of 2 seconds). MS2 spectra were fragmented by HCD (NCE=30) activation mode and the ion-trap was selected as the mass analyzer. The intensity threshold for fragmentation was set to 25,000. A dynamic exclusion of 20 seconds was applied with a mass tolerance of 10 parts per million. Peak lists were generated using Mascot Daemon/Mascot Distiller (Matrix Science, London, England) and imported into the database search program Mascot (version 2.6.0, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances ± 0.5 Da; Met (O) carboxyamidomethyl-Cys specified as variable modification, enzyme specificity was trypsin, with 1 missed cleavage possible. Peaks were searched against the reference genome of the central bearded dragon  and a non-redundant protein database from NCBI (Jan 2015). Raw peak data were imported into Scaffold (Matrix Science, London, England) and analysed accordingly with default settings.
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