Easy nlc liquid chromatograph

LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) coupled to Easy nLC liquid chromatograph (Thermo Scientific) for 60 min. For Nano-LC-MS/MS analysis, the peptide mixture (5 μg) was loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm x 2 cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% formic acid) and separated with buffer B (84% acetonitrile, 0.1% formic acid) in linear gradients at 300 nl/min: 0–35% buffer B for 50 min, 35–100% buffer B for 5 min, and 100% buffer B for 5 min. The mass spectrometer was operated in positive ion mode. The MS data was collected with a data-dependent top10 method by dynamically acquiring the most abundant precursor ions from the survey scan (300–1800 m/z) of higher energy collisional dissociation (HCD) fragmentation. Automatic gain control (AGC) target was set at 3e6, maximum injection time at 10 ms, and dynamic exclusion duration at 40.0 s. Survey scans were acquired at a resolution of 70,000 at 200 m/z, resolution for HCD spectra at 17,500 at 200 m/z, and isolation width at 2 m/z. Normalized collision energy was defined as 30 eV and the underfill ratio as 0.1%. The instrument was run in peptide recognition mode.

The following apparatus were used. Easy-nLC Liquid Chromatograph (Thermo Scientific), Q Exactive Mass Spectrometer (Thermo Scientific), AKTA Purifier 100 (GE Healthcare), Multiscan FC Microplate Photometer (Thermo Scientific), Centrifuges (Eppendorf 5430R), Concentrator plus/Vacufuge (Eppendorf Concentrator Plus) Electrophoresis apparatus (GE Healthcare EPS601), Vertical Electrophoresis Tanks (SE260; GE-Healthcare), Thermofinnigan Easy-nLC 1000, Trap column, ASY column SC001 traps (RP-C₁₈), Analysis column, EASY column SC200 (RP-C₁₈), Maxquant (version 1.3.0.5), Perseus (version 1.3.0.4), MP Fastprep-24 Homogenate instrument (MP Biomedicals), Ultrasonic Cell Disrupter System, Constant temperature incubator, Vortex oscillator, ProteomeDiscoverer 1.4 (Thermo Scientific), MASCOT 2.2 (Matrix Science), Perseus 1.3 (M&M), 10 kDa Ultrafiltration centrifuge tubes, C18 Cartridge, Multiple Affinity Removal LC Column—Human 14/Mouse 3, iTRAQ Reagent‐4/8plex Multiplex Kit, Dissolution buffer (AB SCIEX), SCX chromatographic column, Polysulfoethyl (PolyLCInc, Maryland, U.S.A.).

The LC-MS/MS analysis was conducted on an Easy nLC Liquid Chromatograph (Thermo Fisher Scientific) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific) for 120 min. Next, the peptides were desalted on C18 Cartridges (Empore™ SPE Cartridges C18, bed I.D. 7 mm, volume 3 mL, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) formic acid. Peptides were separated on a C18-reversed phase analytical column (Thermo Scientific Easy Column; 10 cm length, 75 μm inner diameter, 3-μm resin) over a 120 min gradient from buffer A (2% acetonitrile and 0.1% formic acid, vol/vol) and B linear gradient solvent (84% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min controlled by IntelliFlow technology. Data-dependent acquisition was performed with MS scan mass window set at 300−1800 m/z, and top 10 charge state ions were selected for fragmentation. Dynamic exclusion time was set to 50 s. Survey scans were acquired at 17,500 with a maximum ion injection time at 200 m/z. The normalized collision energy was 30 EV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at the maximum fill time, was defined as 0.1%. The instrument was run with the peptide recognition mode enabled. Each sample was analyzed in triplicate.