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Gel-free mass spectrometry analysis of Drosophila melanogaster heads
Here we describe a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on a complete solubilisation and digestion of proteins. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinual sucrose gradient, followed by solubilisation using FASP method, tryptic and consequential chymotryptic digestion of proteins. Peptides were separated by reversed-phase (RP) LC at high pH in the first dimension and acidic RP-LC coupled directly to Orbitrap Velos Pro mass spectrometer.
Sample Processing Protocol
Heads were isolated from 4-5 day old flies using a modification of the standard freezing protocol. Heads were ground into a fine powder with a prechilled mortar and pestle. The powder was re-suspended in ice-cold homogenisation buffer (10 mM HEPES, pH 7.5, 300 mM sucrose, protease inhibitor (Roche Molecular Biochemicals, Mannheim, Germany) and homogenized by Ultra-Turrax (IKA, Staufen, Germany). The homogenate was centrifuged for 10 min at 1,000 x g and the supernatant was centrifuged at 50,000 x g for 30 min. Subsequently, the pellet was re-suspended in washing buffer (homogenization buffer without sucrose), kept on ice for 30 min and centrifuged at 50,000 x g for 30 min. The pellet re-suspended in washing buffer was layered on top of the sucrose cushion (1M and 1,25M sucrose solution) followed by centrifugation at 70,000 x g for 2 h. After centrifugation, fraction from sucrose interface was collected and diluted ten times with washing buffer and then centrifuged at 4°C at 100,000 x g for 30 min. After discarding the supernatant the pellet was stored at 80°C until use. Membrane protein extraction was carried out according to previous study with some modifications […]. Membrane samples were dissolved in a strong-chaotropic extraction buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT and 50 mM Tris-HCl buffer pH 7.5 1 and sonicated for 80 s at 200 cycles . Protein quantities was estimated with the Pierce 660 protein assay (ThermoFisher Scientific, IL, USA).
Data Processing Protocol
LC-MS/MS data were analysed by searching the UniProtKB Drosophila melanogaster database (41,965 sequences, download on January 16th, 2015) with Mascot 2.4 (MatrixScience, London, UK). Detailed search criteria were used as follows: enzyme: trypsin or trypchymo ; cysteine modification: carbamidomethylation; variable modification: oxidation(M); search mode: MS/MS ion search with decoy database search included; peptide mass tolerance ± 10 ppm; MS/MS mass tolerance ± 0.5 Da; acceptance parameters: p < 0.05; ion score cut off: 20.
Aradska J, Bulat T, Sialana FJ, Birner-Gruenberger R, Erich B, Lubec G. Gel-free mass spectrometry analysis of Drosophila melanogaster heads. Proteomics. 2015 Jul 23 PubMed: 26201256
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||43535||no assay title provided (mzIdentML)||1397||4912||2503||22510||3336||
|2||43536||no assay title provided (mzIdentML)||1146||2976||1644||22615||2091||
|3||43489||no assay title provided (mzIdentML)||1415||4374||2410||22985||3160||
|4||43537||no assay title provided (mzIdentML)||605||1066||722||20874||924||
|5||43488||no assay title provided (mzIdentML)||1422||3836||2212||24275||2808||
|6||43538||no assay title provided (mzIdentML)||378||632||435||19745||589||
|7||43487||no assay title provided (mzIdentML)||1457||4206||2309||24082||3002||
|8||43539||no assay title provided (mzIdentML)||518||918||636||20511||829||
|9||43486||no assay title provided (mzIdentML)||1442||3695||2176||24470||2774||
|10||43530||no assay title provided (mzIdentML)||830||1509||1059||21604||1341||