Project PXD001712

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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.


Jana Aradska, MedUni Wien
Gert Lubec, Dept. of Pediatrics, Medical University of Wien, Austria ( lab head )

Submission Date


Publication Date





Not available

Experiment Type

Shotgun proteomics

Assay count



    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


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# 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