Surveyor ms pump

Mass spectrometric analysis was performed on an ion trap mass spectrometer equipped with an electrospray ion source ESI (LCQ-DECA, Thermo Fischer Scientific, San Jose, CA, USA). The mass spectrometer was coupled online with a and autosampler (Thermo Fischer Scientific, San Jose, CA, USA) and a LC-pump (Surveyor MS Pump, Thermo Fischer Scientific, San Jose, CA, USA). Samples were dissolved in methanol ((25 µg/µL) and 5µL were loaded onto a Waters Symmetry RP-C18 column (150 mm × 1 mm i.d., 100 Å, 3.5 µm). Separation was achieved thermosetting the column at 25 °C with a linear gradient of H₂O + 1% FA and ACN + 1% FA at 50 μL/min. Elution was performed, increasing solvent B from 5% to 15% in 25 min, 25% in 40 min, 30% in 45 min, and 55% in 55 min. Full scan mass spectra were acquired in negative ion mode in the m/z range 150–2000. ESI ion source operated with 220 °C capillary temperature, 30 a.u. sheath gas, −3.5 kV source voltage and −18 V capillary voltage. Mass spectrometric analysis was performed by the data-dependent method with normalized collision energy of 30 a.u. and activation Q set as 0.250. Mass calibration was achieved with a standard mixture of caffeine (Mr 194.1 Da), MRFA peptide (Mr 524.6 Da), and Ultramark (Mr 1621 Da). Data acquisition and data analyses were performed with the Xcalibur v. 1.3 Software.

Metabolic labeling of H292 cells was carried out with normal arginine and lysine or heavier isotopic variants of the two amino acids L-Lysine-HCl [¹³C₆], L-arginine-HCl [¹³C₆, ¹⁵N₄] using Invitrogen's SILAC-Flex Media kit and heavy arginine purchased from Thermo Fisher Scientific. To decrease sample complexity, HSP90 immunoprecipitations were performed on drug treated cells and proteins were separated by 1D-SDS-PAGE. Proteins from gel slices were digested using porcine trypsin and analyzed by LC-MS/MS. Peptides were cleaned up and concentrated using C18 stage tips (Proxeon) and separated using online C18 reverse-phase nanoscale liquid chromatography tandem mass spectrometry on a Surveyor MS pump connected to a LTQ-Orbitrap XL (Thermo Fischer Scientific) using a 2 h linear gradient. Fragmentation of the top 10 peptides in each sample was performed by collision-induced dissociation. Raw MS files from LTQ-orbitrap were analyzed using MaxQuant (version 1.2.2.4) [14] (link). MS/MS spectra were searched against the decoy IPI-human database version 3.68 using the Andromeda search engine. A false discovery rate of 0.01 was used on both the peptide and protein levels.