Orbitrap q exactive hf x

The peptides were analyzed on an Orbitrap Q Exactive HF-X (Thermo Fisher Scientific) mass spectrometer coupled to an UltiMate^TM 3000 RSLCnano system (Thermo Fisher Scientific). A 15-cm-long LC column (i.d. 150 μm) packed with 1.9 μm C18 packing particles was used for peptide separation. The column was pulled using a micropipette puller (P-2000; Sutter Instrument) for preparation of the nano-ESI tips with a ∼5 μm opening. The spray voltage was set at 2.3 kV. Because of the high direct current voltage applied, the operator should stay away from high voltage power to avoid danger. An 80-min gradient of 6–40% buffer B (80% acetonitrile with 0.1% formic acid) was used for peptide elution. MS measurements were performed either in data-dependent acquisition mode. The full MS scans were acquired from m/z 350 to 1550 at a resolution of 120,000 (at an m/z 200) with a target of 3e⁶ charges for the automated gain control and 20 ms maximum injection time. For higher energy collisional dissociation MS/MS scans, the normalized collision energy was set to 27%, the resolution was 15,000 at m/z 200, accumulated for a maximum of 30 ms, or until the automated gain control target of 2e⁴ ions was reached.

Proteomic analysis was performed on an Orbitrap Q Exactive HF-X (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer equipped with a nano-electrospray (nano-ESI) source and a high-pressure nanoflow chromatograph UPLC Ultimate 3000 (Thermo Fisher) equipped with a lab-packed reverse-phase (Kinetex C18, 2.4 μm) column (100 μm × 500 mm). The temperature of the column was thermostatically controlled at 60 °C. Samples were loaded in buffer A (0.1% Formic acid) and eluted with a linear (180 min) gradient of 3 to 55% buffer B (0.1% Formic acid, 80% Acetonitrile) at a flow rate of 220 nL/min. Mass spectrometric data were stored during automatic switching between MS1 scans and up to 16 MS/MS scans (topN method). The target value for MS1 scanning was set to 3 × 10⁶ in the range 390–1400 m/z with a maximum ion injection time of 45 ms and a resolution of 60,000. The precursor ions were isolated at a window width of 1.4 m/z. Precursor ions were fragmented by high-energy dissociation in a C-trap with a normalized collision energy of 30 eV. MS/MS scans were saved with a resolution of 15,000 at 400 m/z and a value of 2 × 10⁵ for target ions with a maximum ion injection time of 50 ms.

LC-MS/MS data was acquired by an Orbitrap Q-Exactive HF-X (Thermo, Bremen) coupled to an Ultimate 3000 capillary flow LC-system. Setup was modified from Thermo Scientific Technical note: 72827. Peptide samples were loaded at 150 μl/min (2% ACN, 0.05% TFA) for 30 sec onto a 5 μm, 0.3 × 5 mm, Acclaim PepMap trapping cartridge (Thermo Scientific). Samples were then eluted onto a pulled emitter analytical column (75 μm ID, 15 cm). The analytical column was “flash-packed” [58 (link)] with C₁₈ Reprosil Pur resin (3 μm) and connected by Nanoviper fittings and a reducing metal union (Valco, Houston, TX). The flowrate of the 15 min gradient was 1.2 μL/min with solvent A: 0.1% formic acid (FA) and solvent B: 0.1% FA in 80% ACN. Gradient conditions for solvent B were as followed: 8% to 25% in 10 min, 25% to 45% in 1.7 min. The trapping cartridge and the analytical column were washed for 1 min at 99%B before returning to initial conditions. The column was equilibrated for 2 min. MS settings: ESI spray voltage 2 kV, cap temp = 275°C, Resolution: 60 k, micro scans = 1, max IT = 100 ms, AGC = 3 × 10⁶, MSMS resolution 15 k, n = top 5, max IT = 100 ms, AGC = 1 × 10⁵.

The tryptic peptide mixture was acidified with formic acid, cleaned with C18 Monospin columns and analyzed on the Orbitrap Q Exactive HF-X (Thermo Scientific). MS/MS was performed using Higher-Energy C-Trap Dissociation (HCD). Mass spectra were analyzed using Byonic v2.14.27 (Protein Metrics) allowing for 12 ppm mass tolerances. Variable post-translational modifications were allowed for, including oxidation, methylation, carbamylation, and phosphorylation. Proteins were limited to those scoring better than a 1% false discovery rate (FDR). Comparisons of protein abundance and MS data were made to select the optimal method for kidney tissue collection between FFPE and OCT.

The peptide mixtures were analyzed by nano-LC–MS/MS on an Orbitrap Q-Exactive HF-X (Thermo Fisher Scientific USA) coupled to an EASY-LC 1000 system (Thermo Fisher Scientific, USA). The data was generated by using previously described protocol³⁰ .

Peptides were loaded onto 50 cm fused silica columns packed in-house (ReproSil Pur C18-AQ, 1.9 μm particle size) and maintained at 60 °C using a column oven (Sonation, GmbH). Peptides were separated by reversed-phase chromatography using an Easy nLC 1200 (Thermo Fisher Scientific), with a binary buffer system of 0.1% formic acid (buffer A) and 80% ACN/0.1% formic (buffer B). Peptides were separated by linear gradients of buffer B (3 to 25% over 90 min, followed by 25-35% over 20 min, and 35-60% over 10 min) at a flow rate of 300 nL/min, and electrosprayed directly into the mass spectrometer by the application of 2.4 kV with a liquid junction union. Ionized peptides were analyzed using a benchtop Orbitrap (Q Exactive HF-X) mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent mode, performing survey scans of 3e⁶ ions at a resolution of 60,000 from 300–1650 m/z. The 15 most abundant precursors from the survey scan with charge state >1 and <5 were selected for fragmentation. Precursors were isolated with a window of 1.4 m/z and fragmented in the HCD cell with NCE of 27. Maximum ion fill times for the MS/MS scans were 28 ms, with a target of 2.9e³ ions (intensity threshold 2.9e⁵ ions). Fragment ions were analyzed with high resolution (15,000) in the Orbitrap mass analyzer. Dynamic exclusion was enabled with duration 30 s.