100 islets from wildtype and islet beta-cell Ins1Cre/+ ; Cpefl/fl mice were pooled and processed for top-down proteomic analysis. Briefly, homogenization buffer (8 M urea, 100 mM ammonium bicarbonate [ABC], 5 mM ethylenediaminetetraacetic acid [EDTA], 1 mM phenylmethylsulfonyl fluoride [PMSF]) was added to each sample before vortexing for 10 sec to resuspend islets followed by sonication in a water bath for 5 min at room temperature (rt). Reduction was accomplished through addition of 14 uL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) with incubation at rt for 2 h within a ThermoMixer (ThermoFisher) set at 1,200 RPM. This was followed by alkylation using 20 uL of 0.5 M iodoacetamide (IAA) and incubation for 30 min at rt in complete darkness at 1,200 RPM. The reaction was quenched through addition of 50 uL of dithiothreitol (DTT) before clarification of samples with 15 min centrifugation at 18,000 RCF at 10 deg C. The resulting supernatant was added to a 3 K MWCO Amicon Ultra 0.5 mL centrifugal filter (MilliporeSigma) and centrifuged at 14,000 RCF for 60 min at 10 deg C. Samples were analyzed using a Waters NanoACQUITY UPLC system with mobile phases consisting of 0.2% FA in H2O (Mobile Phase A) and 0.2% FA in ACN (Mobile Phase B). Both trapping-precolumn (150 um i.d., 5-cm length) and analytical column (100 um i.d., 50-cm length) were slurry-packed with C2 packing material (5 um and 3 um for trap/analytical respectively, 300 Angstrom, Separation Methods Technology). Samples were loaded into a 10 uL loop, corresponding to 400 ng of loaded material, and injected into the trapping column with an isocratic flow of 1% B at 5 uL/min over 15 min for desalting. Separation was performed with a 1% to 50% B gradient over 140 min at 300 nL/min. For MS/MS analysis of proteins, the NanoACQUITY system was coupled to a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with the FAIMS Pro interface.
[doi:10.25345/C5G15TN11]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: FAIMS ; top-down proteomics ; pancreas ; diabetes ; insulin
Principal Investigators: (in alphabetical order) |
Wei-Jun Qian, Pacific Northwest National Laboratory, United States |
Submitting User: | alchemistmatt |
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