Visible bands have been cut in the gel, destained and washed with double-distilled H2O (two occasions for 5 min every single time) and then with 50 ethanol (two instances for five min every time) ahead of being stored at 20 . Gel pieces were tryptically digested as previously described (26) in 25 mM triethylammonium bicarbonate buffer at 37 overnight with 12 ng/ l trypsin (catalog no. V5111; Promega, Madison, WI). For in-solution digestion, P3 samples had been resuspended in 13.2 mM SA (pH three)8 M urea00 mM DTT and incubated for 1 h at RT, followed by 15 min at 70 . Iodoacetamide was added to ten mM, and proteins had been alkylated by incubation for 15 min at RT within the dark. Samples have been added to a Phospholipase Source prerinsed spin filter (Amicon Ultra 30K or 10K device; catalog no. UFC503008/UFC501008; EMD Millipore, Billerica, MA) and centrifuged at 14,000 g (27). Samples have been washed with 9 M urea and after that with 25 mM ammonium bicarbonate. Samples have been digested with 12 ng/ l trypsin in 25 mM ammonium bicarbonate overnight. Right after digestion, samples had been spun at 14,000 g and washed two times with 25 mM ammonium bicarbonate. The retentate was transferred to a brand new tube and air dried. For on-membrane digestion, the samples had been dotted onto 0.1- mpore-size nitrocellulose membrane and digested by trypsin by a process adapted from reference 28. Briefly, P3 samples were resuspended and treated as for in-solution digestion. Samples had been then dotted onto the membrane by gravity. Wells have been rinsed with 20 mM SA (pH three), followed by TBS. Dots had been cut and air dried. Right after protein digestion with 12 ng/ l trypsin in 25 mM ammonium bicarbonate, the membranes had been dissolved with acetone as well as the precipitated peptides had been air dried. All digested peptides have been reconstituted in 2 acetonitrile0.1 formic acid for mass spectrometry (MS) analysis. MS information acquisition. Protein identification by Dopamine Receptor MedChemExpress liquid chromatography-tandem MS (LC-MS/MS) analysis of peptides was performed with an LTQ Orbitrap Velos MS (Thermo Scientific) interfaced with a 2D nanoLC method (Eksigent, Dublin, CA). Peptides had been fractionated by reversephase high-performance liquid chromatography on a PicoFrit column (75 m by 10 cm) with a 15- m emitter (catalog no. PF3360-75-15-N-5; New Objective, Woburn, MA) packed in property with Magic C18AQ (5 m, 120 Michrom Bioresources, Inc., Auburn, CA) using a 1 to 45 acetonitrile0.1 formic acid gradient more than 60 min at 300 nl/min. Eluting peptides were sprayed directly into an LTQ Orbitrap Velos at two.0 kV. Survey scans (full MS) had been acquired from 350 to 1,800 m/z with as much as ten peptide masses (precursor ions) individually isolated with a 1.two Da window and fragmented (MS/MS) with a collision energy of HCD35, 30 s dynamic exclusion. Precursor and fragment ions had been analyzed at 30,000 and 15,000 resolution, respectively. Protein and peptide identification. MS/MS spectra have been extracted together with the ProteoWizard Toolkit (29). The spectra had been analyzed together with the GPM Manager (version 2.two.1) and X!Tandem (30) to search against a homemade mouse database containing 213,054 nonredundant protein sequences produced with mouse sequences in the Ensembl database (files Mus_musculus.GRCm38.73.pep.all and Mus_musculus.GRCm38.73. pep.abinitio) and from the NCBI database (file nr downloaded on 09/19/ 2013) as described in reference 16. Two searches had been performed by using completely or semitryptic enzyme specificity (see deposited MS data for facts). Peptides and proteins which have an expectation worth of log10 (e) two have been incorporated.
