Computational protocol: Distinct Regulation of Transmitter Release at the Drosophila NMJ by Different Isoforms of nemy

Similar protocols

Protocol publication

[…] The CNS of 50 third instar larvae were dissected and crushed in 40 ul of an ice-cold solution of methanol/water/formic acid (90:9:1) solution, filtered through Millipore spin-down filters (0.22mm, 6000 rpm for 10 min) dried and stored at -20°C. The pellets were resuspended in 5% acetonitrile and 0.1% formic acid. The peptides were separated by LCMS on a reverse phase column (Magic C18, Michrome Biosciences) using a split-free nano chromatography system (EASY-nLC, Proxeon, Odense Denmark). Samples were loaded via a C18 pre-column and analysis was carried out in duplicate. Elution was performed across a gradient from 5% to 30% acetonitrile in 0.1% Trifluoroacetic acid over 50 min. Peptide detection was carried out using a linear ion trap/FTMS hybrid mass spectrometer (LTQ-Orbitrap, Thermo-Fisher, Bremen Germany) operated in a data dependent mode. Peptide MS data was collected at a resolution of 60,000 full-width half maximum. Tandem mass spectra were obtained in parallel in the lineAr ion trap. The tandem mass spectra were compared to the D. melanogaster database (NCBI) using the Sequest search engine (Thermo-Fisher, San Jose CA). Potential modifications included in the search were amidated C-termini and oxidized methionine residues. The mass accuracy used in the search was 7 ppm for the parent ion and 0.5 Daltons for the fragment ions. Peptide quantitation was carried out using the Xcalibur 2.0 software (Thermo-Fisher, San Jose CA) generated extracted ion current for the monoisotopic isotopic peak of the doubly charged ion. The total area of the extracted peak for each ion was calculated for all samples. All measurements were done in triplicate biological replicates each of which contained two technical repetitions. [...] Data were analysed statistically using an ANOVA and t-test. All statistics were performed using Excel and Sigmaplot software. […]

Pipeline specifications

Software tools Comet, SigmaPlot
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Drosophila melanogaster
Chemicals Glycine, Tyramine