Rnd2 No Longer A Hidden working experience
Peptide mixtures concentration was estimated by measuring Rnd2 absorbance at 280?nm as described above. In solution digestion (ISD) Protein extracts were diluted with Milli-Q water to a final 0.2% SDS concentration, then dispensed on the top of Detergent Removal Spin Columns (Pierce, Rockford, IL, USA), incubated for 2?min at room temperature, and centrifuged for 2?min at 1,500 �� g, according to the manufacturer��s instructions. The eluted solution was then sequentially incubated for 30?min at 56��C in 10?mM DTT, 50?mM ABC for reduction and for 30?min at room temperature in the dark in 25?mM IAM, 50?mM ABC for alkylation. Trypsin digestion was performed overnight at 37��C (enzyme-to-protein ratio 1:100 w/w). The digestion was stopped by adding 10?��l of 20% TFA and the solution was brought to dryness. Finally, the peptide mixture was resuspended in 0.2% formic acid to an approximate final concentration of 1?mg/ml. LC-MS/MS analysis MS analysis was carried out using an LTQ-Orbitrap Velos (Thermo Scientific) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA, now part of Thermo Scientific). Before loading, peptide mixtures were purified using ZipTip Pipette Tips (Millipore), according to the manufacturer��s recommendations. After loading, peptide mixtures (4?��g per run) were concentrated and desalted on a trapping pre-column (Acclaim PepMap C18, 75?��m��2?cm nanoViper, 3?��m, 100??, Thermo Selleckchem FK866 Scientific), using 0.2% formic acid at a flow rate of 5?��l/min. The peptide separation was performed at 35��C using a C18 column (Acclaim PepMap RSLC C18, 75?��m �� 15?cm nanoViper, 2?��m, 100??, Thermo Scientific) at a flow rate of 300?nL/min, using a 485?min gradient from 1 to 50% eluent B (0.2% formic acid in 95% ACN) in eluent A (0.2% formic acid in 5% ACN). The mass spectrometer LTQ-Orbitrap Velos was set up in a data dependent MS/MS mode, as described previously [48]. Briefly, the lock mass option was enabled on a protonated polydimethylsiloxane background ion for internal recalibration, peptide ions were selected as the ten most intense peaks of the previous scan, and Higher Energy selleckchem Collisional Dissociation (HCD) was chosen as the fragmentation method. Data analysis Protein identification was performed using Proteome Discoverer (version 1.4.0.288; Thermo Scientific), with a workflow consisting of the following nodes (and respective parameters): Spectrum Selector for spectra pre-processing (precursor mass range: 350�C5000?Da; S/N Threshold: 1.5), Sequest-HT as search engine (Protein Database: Homo sapiens sequences from UniProtKB/SwissProt, release 2013_12; Enzyme: Trypsin; Max. missed cleavage sites: 2; Peptide length range 5�C50 amino acids; Max. Delta Cn: 0.05; Precursor mass tolerance: 10?ppm; Fragment mass tolerance: 0.02?Da; Static modification: cysteine carbamidomethylation; Dynamic modification: methionine oxidation), and Percolator for peptide validation (FDR
