MassIVE MSV000085627

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Drosophila indirect flight muscle

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Description

LC-MS/MS analysis of peptide digests of myofibrils from wild type and Dmlc2(Delta 2-46; S66A, S67A) mutant Drosophila flies. Myofibrils and thick filament samples were solubilized in 150 ul 0.1% RapiGest SF Surfactant (Waters Corporation; 50 C, 1 hour). Proteins were reduced by addition of 0.75 ul of 1M dithiothreitol and heating (100 C, 10 min). Cysteines were alkylated by addition of 22.5 ul of 100 mM iodoacetamide in 50 mM ammonium bicarbonate and incubation in the dark (22C, 30 min). Proteins were digested to peptides by addition of 25 ul of 50 mM ammonium bicarbonate containing 5 ug of trypsin (Promega) and incubating (37 C, 18 hours). The samples were dried down in speed vacuum device. Trypsin was deactivated and RapiGest was cleaved by addition of 100 ul of 7% formic acid in 50 mM ammonium bicarbonate and heating (37 C, 1 hour). The resultant peptides were dried down. RapiGest was cleaved again by addition of 100 ul of 0.1% trifluoroacetic acid and heating (37 C, 1 hour). The resultant peptides were dried down and reconstituted a final time in 60 ul of 0.1% trifluoroacetic acid. The samples were centrifuged at 18,800 RCF for 5 minutes (Thermo, Sorvall Legend Micro 21R) to pellet the surfactant. The top 57 ul of solution was transferred into a mass spectrometry (MS) analysis vial. A 20 ul aliquot of each sample was injected onto an Acquity UPLC HSS T3 column (100 A, 1.8 um, 1 x 150 mm) (Waters Corporation) attached to a UltiMate 3000 ultra-high pressure liquid chromatography (UHPLC) system (Dionex). The peptides were separated and the UHPLC effluent was directly infused into a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (MS) through an electrospray ionization source (Thermo Fisher Scientific). Data were collected in data-dependent MS/MS mode with the top five most abundant ions being selected for fragmentation. [doi:10.25345/C52H8B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Drosophila, indirect flight muscle, Dmlc2(Delta 2-46 ; S66A, S67A), thick filament

Contact

Principal Investigators:
(in alphabetical order) 		Michael Previs, University of Vermont, United States
Submitting User: mprevis
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