Project PXD011171

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Biological Dataset
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Summary

Title

Central bearded dragon hibernation label-free LC-MS/MS

Description

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 [14] 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.

Contact

Alexander Capraro, UNSW Australia
Paul D Waters, Waters Lab, School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Australia, Australia ( lab head )

Submission Date

24/09/2018

Publication Date

18/04/2019

Software

Not available

Modification

Carbamidomethyl
Oxidation

Quantification

Label free

Experiment Type

Shotgun proteomics

Assay count

18

Publication

Publication pending

Assay

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Showing 1 - 10 of 18 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 99218 AC-1-21-2-17-FLIT-60min-1ul.raw (F135381).mzid 2373 8138 2813 3666 0
2 99229 AC-2-21-2-17-FLIT-60min-1ul.raw (F135382).mzid 3051 10376 3939 5054 0
3 99217 AC-3-21-2-17-FLIT-60min-1ul.raw (F135383).mzid 2367 7791 2868 3732 0
4 99228 AC-4-21-2-17-FLIT-60min-1ul.raw (F135378).mzid 2616 8521 3136 3994 0
5 99219 AC-5-21-2-17-FLIT-60min-1ul.raw (F135379).mzid 3019 10773 3759 4893 0
6 99225 AC-6-21-2-17-FLIT-60min-1ul.raw (F135380).mzid 2465 9390 3365 4545 0
7 99224 AC-10-21-2-17-FLIT-60min-1ul_r.raw (F135443_1).mzid 1836 7581 2489 3340 0
8 99216 AC-11-21-2-17-FLIT-60min-1ul_r.raw (F135442_1).mzid 1750 7138 2187 2893 0
9 99227 AC-12-21-2-17-FLIT-60min-1ul.raw (F135390).mzid 2215 8672 3009 3994 0
10 99215 AC-7-21-2-17-FLIT-60min-1ul_r.raw (F135441).mzid 2136 9828 2892 3831 0