The dried gel pieces were rehydrated by adding 10 μL of ammonium bicarbonate buffer (50 mM) containing trypsin (20 ng/μL; Promega Trypsin Gold) and incubated for 16 h at 37 °C to ensure efficient peptide digestion. Gel pieces were washed with 30 μL of formic acid (5%, v/v) in acetonitrile (50% v/v) for 30 min. This step was repeated twice for complete peptide removal. Digestion solutions were pooled in low-retention micro tubes and the volume was reduced to approximately 10 μL by vacuum centrifugation. The samples Torin 1 manufacturer were desalted by reversed phase chromatography (Zip tips, C18 Ultra-Micro Prep Tip, Millipore Corporation, Bedford, MA). Briefly,

the Zip tips were initially washed three times with 10 μL 0.1% trifluoroacetic acid (TFA)/60% ACN and rinsed three times with 10 μL of 0.1% TFA. Then samples were loaded by aspiration before being eluted with 60% ACN/0.1% TFA. Tryptic digests, obtained as described previously, were submitted to reversed-phase nanochromatography coupled to nanoelectrospray high resolution mass spectrometry for identification. Four microliters of desalted tryptic peptide digest were initially applied to a 2 cm long (100 μm internal diameter) trap column packed with 5 μm, 200 A Magic C18 AQ matrix (Michrom Bioresources, USA) followed by separation on a 10 cm long (75 μm internal diameter) separation column that was packed with

the same matrix, MK-2206 supplier directly on a self-pack 15 μm PicoFrit empty column (New Objective, USA). Chromatography was carried out on an EASY-nLC II instrument (Thermo Scientific, USA). Samples were loaded onto the trap column at 2000 nL/min while chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% (v/v) formic acid in water while mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile and gradient conditions were as follows: 2–40% B in 32 min; up to 80% B in

4 min, maintaining at this concentration for 2 min more, before column reequilibration. Eluted peptides were directly introduced to an LTQ XL/Orbitrap mass spectrometer (Thermo, USA) for analysis. For each spectra, selleck kinase inhibitor the 10 most intense ions were submitted to CID fragmentation followed by MS2 acquisition on the linear trap analyzer. Uninterpreted tandem mass spectra were searched against the no redundant protein sequence database from the National Center for Biotechnology Information (NCBI) using the Peaks Client 5.3 build 20110708. The search parameters were as follows: metazoan taxon, no restriction of protein molecular weight, two missed trypsin cleavage allowed, non-fixed modifications of methionine (oxidation) and cysteine (carbamidomethylation) with no other post-translational modifications being taken into account. Mass tolerance for the peptides in the searches was 10 ppm for MS spectra and 0.6 Da for MS/MS.