Q exactive hf x quadrupole orbitrap mass spectrometer

Samples were eluted from StageTips with 50 μl buffer B (80% acetonitrile (ACN) and 0.5% acetic acid), the organic solvent was removed in a SpeedVac concentrator for 20 min, and peptides were resuspended in 10 μl of Buffer A (2% ACN and 0.1% TFA). 3 μl of each sample was analyzed by nanoflow liquid chromatography on an EASY-nLC system (Thermo Fisher Scientific, Bremen, Germany) on-line coupled to an Q Exactive HF-X quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific) through a nanoelectrospray ion source (Thermo Fisher Scientific). A 50 cm column with 75 μm inner diameter was used for the chromatography, in-house packed with 3 μm reversed-phase silica beads (ReproSil-Pur C₁₈-AQ, Dr. Maisch GmbH, Germany). Peptides were separated and directly electrosprayed into the mass spectrometer using a linear gradient from 5% to 60% ACN in 0.5% acetic acid over 120 min at a constant flow of 300 nl/min. The linear gradient was followed by a washout with up to 95% ACN to clean the column for the next run. The overall gradient length was 145 min. The QExactive HF-X was operated in a data-dependent mode, switching automatically between one full-scan and subsequent MS/MS scans of the fifteen most abundant peaks (Top15 method), with full-scans (m/z 300–1650) acquired in the Orbitrap analyzer with a resolution of 60,000 at 100 m/z.

Peptides were resuspended in water with 0.1 % formic acid (FA) and analyzed using EASY-nLC 1200 nano-UHPLC coupled to Q Exactive HF-X Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The chromatography column consisted of a 40 cm long, 75 μm i.d. microcapillary capped by a 5 μm tip and packed with ReproSil-Pur 120 C18-AQ 2.4 μm beads (Dr. Maisch GmbH). LC solvents were 0.1 % FA in H₂O (Buffer A) and 0.1 % FA in 90 % MeCN: 10 % H₂O (Buffer B). Peptides were eluted into the mass spectrometer at a flow rate of 300 nL/min. over a 240 min. linear gradient (5–35 % Buffer B) at 65°C. Data were acquired in data-dependent mode (top-20, NCE 28, R = 7’500) after full MS scan (R = 60’000, m/z 400–1’300). Dynamic exclusion was set to 10 s, peptide match to prefer and isotope exclusion was enabled.

Peptides were resuspended in water with 0.1% formic acid (FA) and analyzed using EASY-nLC 1200 nano-UHPLC coupled to Q Exactive HF-X Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The chromatography column consisted of a 50 cm long, 75 μm i.d. microcapillary capped by a 5 μm tip and packed with ReproSil-Pur 120 C18-AQ 2.4 μm beads (Dr. Maisch GmbH). LC solvents were 0.1% FA in H₂O (Buffer A) and 0.1% FA in 90% MeCN: 10% H₂O (Buffer B). Peptides were eluted into the mass spectrometer at a flow rate of 300 nL/min over a 240 min linear gradient (5-35% Buffer B) at 65°C. Data were acquired in data-dependent mode (top-20, NCE 28, R = 7,500) after full MS scan (R = 60,000, m/z 400-1,300). Dynamic exclusion was set to 10 s, peptide match to prefer, and isotope exclusion was enabled.