Eksigent nanolc ultra 2d plus system

Buffer A (0.1% formic acid, 2% ACN) was added to dried peptides. Mass spectrometry was performed with an Eksigent nanoLC Ultra 2D plus system (AB SCIEX, CA) coupled with a Triple TOF 5600 System (AB SCIEX, MA). The peptide samples underwent injection into a C18 nanoLC trap column (C18, 100 μm × 3 cm, 3 μm, 150 Å) and were washed using 2% ACN (0.1% formic acid) for 10 min at 2 μL/min. Peptides were eluted using a gradient of 5–35% ACN (0.1% FA) over 70 min with an analytical ChromXP C18 column (C18, 75 μm × 15 cm, 3 μm 120 Å) with a spray tip, and were then introduced into the mass spectrometer which was fitted with a NanoSpray III source (AB SCIEX, Canada). This mass spectrometer operated in a data-dependent manner. A cyclic series of a full mass spectrometry scans was then acquired in 250 ms periods, with a dynamic exclusion of 18 s. The total cycle time was fixed at 2.5 s. Collision-induced dissociation was performed using a rolling collision energy setting.

Labeled peptides were analyzed via LC-MS/MS with an LTQ-Orbitrap Velos (Thermo-Fisher Scientific) interfaced with an Eksigent nanoLC ultra 2D plus system (AB Sciex, Switzerland). Peptides were injected onto a Pepmap 100 trap column (300 μm × 5 mm, 5 μm, 100 Å) at a flow rate of 400 nL/min. For analytical separation, the trap was switched inline to an Acclaim Pepmap C18 column (75 μm × 15 cm, 3 μm, 100 Å) using a 240 min linear gradient from 5 to 30% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min. MS/MS analyses were performed using an LTQ Orbitrap Velos (Thermo Fisher Scientific) with a nanoelectrospray ion source. The LTQ-Orbitrap Velos settings included one 30,000 resolution scan for precursor ions followed by MS2 scans of the 20 most intense precursor ions in positive ion mode. MS/MS data acquisition was completed using Xcalibur 2.1 (Thermo Fisher Scientific). The fragmentation method used for identification of TMT labeled peptides was based on higher energy collisional dissociation (HCD) with 40% fixed collision energy (CE).

Samples were subjected to an Eksigent Nano LC ultra 2D plus system (AB Sciex, Waldbronn, Germany) connected to Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB Sciex, Waldbronn, Germany). The calibration of system was done using beta-galactosidase and combined with C18 trap column and C18 RP analytical column for the analysis of digested peptide fragments. Sample trapping and washing was carried out at a flow of 5 μl in 12 min run time with 100% solvent A (water +0.1% formic acid), and elution was accomplished during a 45 min. gradient from 13% to 32% Solvent B (ACN +0.1% formic acid) at 550 nL/min. The positive ion mode was selected for running the sample, with MS window from 350 to 1250 m/z. Parent ions were fragmented by Atmospheric Pressure Ionization. The flow rate was split less and the column flow through was directly introduced into the Nano spray III ion source. MS-MS data were extracted for database searches.