Peptides were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) using the EASY-nLCnano-LC system (Thermo Fisher Scientific, Bremen, Germany) with a self-packed column (75 μm × 150 mm; 3 μm ReproSil-Pur C18 beads, 120 Å, Dr Maisch GmbH, Ammerbuch, Germany) at a flow rate of 300 nL/min. The RP-HPLC mobile phase A was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. Peptides were eluted over a 90 min period using a gradient (2–90% mobile phase B) into a nano-ESI orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent mode with each full MS scan (m/z 300–1500) followed by MS/MS for the 12 most intense ions with following parameters: ≥+2 precursor ion charge, 2 Da precursor ion isolation window, 80 first mass, and 38 normalized collision energy of HCD. Dynamic Exclusion™ was set for 30 s. The full mass and the subsequent MS/MS analyses were scanned using an Orbitrap analyser with R = 60,000 and R = 15,000, respectively.
Nano esi orbitrap elite mass spectrometer
Manufactured by Thermo Fisher Scientific
The Nano-ESI orbitrap Elite mass spectrometer is a high-resolution, high-mass accuracy instrument that utilizes Orbitrap technology for mass analysis. It is designed to provide sensitive and precise measurements of molecular masses.
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2 protocols using nano esi orbitrap elite mass spectrometer
1
Peptide Separation and Identification by RP-HPLC-MS/MS
2
Nano-LC-MS/MS Proteomic Analysis
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The reverse phase high-performance liquid chromatography (RP-HPLC) separation was achieved on the Easy nano-LC system (Thermo Fisher Scientific) using a self-packed column (75 μm × 150 mm; 3 μm ReproSil-Pur C18 beads, 120 Å, Dr. Maisch GmbH, Ammerbuch, Germany) at a flow rate of 300 nL/min. The mobile phase A of RP-HPLC was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. The peptides were eluted using a gradient (2–90% mobile phase B) over a 90 min period into a nano-ESI Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent mode with each full MS scan (m/z 300–1,500) followed by MS/MS for the 12 most intense ions with the parameters: ≥ +2 precursor ion charge, 2 Da precursor ion isolation window, 80 first mass and 38 normalized collision energy of HCD. Dynamic Exclusion™ was set for 30 s. The full mass and the subsequent MS/MS analyses were scanned in the Orbitrap analyzer with R = 60,000 and R = 15,000, respectively.
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