The 10 fractions were analyzed by LC-MS/MS using an UltiMate 3000 RSLCnano system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The TMT-labeled peptides were loaded on a PepMap 100 C18 precolumn (300 μm × 5 mm; 5-μm particle size; Thermo Fisher Scientific) at 10 μL/min and then transferred to an analytical pulled-emitter column (50 cm × 75 μm), home packed with Inertsil ODS-3 2-μm sorbent (GL Sciences).62 (link) Peptides were separated with a flow rate of 450 nL/min at 45°C in a home-made column thermostat using a gradient from 4% to 32% buffer B in 2 h (where buffer A was 0.1% [v/v] formic acid in water and buffer B was acetonitrile with 0.1% [v/v] formic acid). MS analysis was carried out in data-dependent acquisition (DDA) mode with survey FTMS1 spectra recorded using a target resolution of 120,000, AGC target of 3e6, maximum injection time of 100 ms, scan range from m/z 300 to 2,000, followed by 20 FTMS2 spectra recorded with a target resolution of 60,000, AGC target of 2e5, maximum injection time of 108 ms, quadrupole isolation window of 1.2, fixed first mass at m/z 110, collision energy at 30%, and dynamic exclusion for 40 s.
Pepmap 100 c18 precolumn
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The PepMap 100 C18 precolumn is a reversed-phase HPLC column designed for sample clean-up and pre-concentration prior to LC-MS analysis. It features a 100 Å pore size and 3 µm particle size C18 stationary phase.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Pepmap 100 c18 analytical column, by Thermo Fisher Scientific (1 mentions)
Easy nanolc 1200 instrument, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
2 protocols using pepmap 100 c18 precolumn
1
Comprehensive Proteomic Analysis by LC-MS/MS
2
Peptide Profiling using Orbitrap Fusion Lumos
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Each peptide sample underwent triplicate LC−MS/MS runs using an Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, Germany) coupled with an online EASY-nanoLC™ 1200 instrument (Thermo Fisher Scientific, Germany). Samples were first loaded onto a 75 μm × 2 cm nanoViper PepMap™100 C18 precolumn and then separated on a 75 μm × 50 cm nanoViper PepMap™100 C18 analytical column (Thermo Fisher Scientific, Germany). Mobile phase consisted of 0.1% FA (A) and 0.1% FA/80% ACN (B). The gradient profile (240 min) was set as follows: 3–7% B for 2 min, 7–35% B for 166 min, 35–68% B for 40 min, 68–99% B for 10 min, and 99% B for 22 min. The parameters of mass spectrometry were set as follows: for MS1, scan range of orbitrap spectra (automatic gain control AGC 4 × 105) were from 350 to 1,800 m/z at a resolution of 60 K. For MS2, the multiply charged ions were fragmented in the collision cell by higher-energy collisional dissociation (HCD, collision energy 30%) with an isolation window of 1.6 m/z, a maximum injection time of 30 ms, a resolution of 15 K and AGC target of 5 × 104.
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