Q exactive hf mass spectrometer ms

Samples were analyzed on an Ultimate 3000 rapid-separation liquid chromatography nano (RSLCnano) high-performance LC (HPLC) system coupled with a Q Exactive HF mass spectrometer (MS; both from Thermo Scientific). The HPLC was equipped with a trapping column (100 μm by 2 cm, C₁₈, PepMap RSLC; Thermo Scientific) for preconcentration and an analytical column (75 μm by 50 cm C₁₈, PepMap RSLC; Thermo Scientific) for separation of the peptides. Preconcentration was performed for 5 min at a flow rate of 20 μl/min using 0.1% trifluoroacetic acid (TFA), and separation was performed at a flow rate of 250 nl/min. An optimized binary gradient of solvent A (0.1% formic acid [FA]) and solvent B (84% acetonitrile, 0.1% FA) was used with the following steps: 0 min, 2% B; 5 min, 2% B; 10 min, 5% B; 50 min, 9% B; 73 min, 15% B; 100 min, 21% B; and 115 min, 45% B. This was followed by two washing steps for 5 min at 95% B and 20 min of equilibration at 2% B. The MS was operated in PRM mode at a resolution of 60,000 (at 200 m/z) with a fixed first mass of 150 m/z. The AGC target was set to 1 × 10⁶ and a maximum injection time of 118 ms. Targeted precursors were isolated with a quadrupole isolation width of 0.4 m/z and fragmented with a normalized collision energy of 27. PRM acquisition was scheduled with a retention time window of 2 min per target.

A Q-Exactive HF mass spectrometer (MS) (Thermo Scientific, USA) was used within LC–MS/MS study. Thermo Scientific, USA’s precolumn was used to enrich and desalt 5 ul of the peptide before separating it at a 300 ml/min flow rate for 120 min through the C18 column having 2–40% gradient mobile phase B. The main mass spectrometer’s resolution was 60,000, and its scanning range was 300–1800 m/z. The MS was run within top-ranking 20 (data-dependent acquisition method) mode. Twenty parent-ions having greatest signals were chosen for secondary mass spectrometry scans after finishing the first mass spectrometry. The dynamic exclusion was set at 30 s, the resolution was 15,000, and the fragmentation mode was HCD. MaxQuant (v1.4.1.2, http://www.maxquant.org/) searched each sample raw data file and identify and quantify any observable characteristics.