Q exactive hf x hybrid quadrupole orbitrap mass spectrometer

Chromatographic and HRMS data were acquired on a Vanquish UHPLC system coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) using conditions according to published untargeted metabolomics methods [11 (link),12 (link),13 (link),14 (link),15 (link),16 (link)]. Chromatographic data were acquired using an HSS T3 C18 column (2.1 × 100 mm, 1.7 µm, Waters Corporation, Milford, MA, USA) at 50 °C with binary mobile phases of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The linear gradient consisted of an initial composition of 2% B, increased to 100% B over 16 min, and was held at 100% B for 4 min, with a flow rate at 0.4 mL/min. Data-dependent acquisition was used to acquire spectral data from 70 to 1050 m/z. The untargeted data were then processed using Progenesis QI (Waters Corporation). Data were filtered by removing peaks with a higher average abundance in blank injections as compared to QCSP injections. Peaks were normalized using the “Normalize to All” function in Progenesis except for the TMA samples, which were normalized to the total intensity.

Metabolomics data were acquired via previously published UHPLC-HRMS methods using a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an HSS T3 C18 column (2.1 × 100 mm, 1.7 µm, Waters Corporation) held at 50 °C [91 (link),92 (link),93 (link),94 (link),95 (link),96 (link),97 (link),98 (link)]. A binary pump was used with water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B) as mobile phases. The mobile phase gradient started from 2% B, increased to 100% B in 16 min, and was then held for 4 min with a flow rate of 400 µL/min. Mass spectral data were collected using a data-dependent acquisition mode in positive polarity at 70–1050 m/z. QCSP and blank injections were placed at a rate of 10% throughout the study samples. An injection volume of 5 µL was used for analysis of each sample. Raw UHPLC-HRMS data were imported into Progenesis QI (version 2.1, Waters Corporation, MA, USA) for alignment, peak picking, and deconvolution. Background signals were removed by filtering out peaks with a higher average abundance in the blank injections as compared to the QCSP injections. Data were normalized using a QCSP reference sample using the “normalize to all” function in progenesis [99 (link)].

Metabolomics data were acquired via previously published UHPLC-HRMS methods [26 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 (link)]. The analysis utilized a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an HSS T3 C18 column (2.1 mm × 100 mm, 1.7 µm, Waters Corporation) held at 50 °C. A binary pump was used with water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B) as mobile phases. The mobile phase gradient started from 2% B, increased to 100% B in 16 min, and was then held for 4 min with a flow rate of 400 µL/min. Mass spectral data were collected using a data-dependent acquisition mode in positive polarity at 70–1050 m/z. QCSP and blank injections were placed at a rate of 10% throughout the study samples. An injection volume of 5 µL was used for analysis of each sample. Raw UHPLC-HRMS data were imported into Progenesis QI (version 2.1, Waters Corporation, MA, USA) for alignment, peak picking, and deconvolution. Background signals were removed by filtering out peaks with a higher average abundance in the blank injections as compared to the QCSP injections. Data were normalized using a QCSP reference sample using the “normalize to all” function in progenesis [38 (link)].

All synthetic peptides were obtained from Genscript as a library service and crude purity. We measured the peptide libraries and HLA-peptides by LC-MS on an Ultimate 3000 RSLCnano System coupled with an Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). Peptides were loaded onto the analytical column (PepMap C18 column, 2 µm particle size, 75 µm × 50 cm; Thermo Scientific) and eluted in a 60 min linear gradient from 3% to 25% ACN in 1% DMSO/0.1% formic acid at a flow rate of 250 nl/min. Peptides were introduced to the mass spectrometer using an EasySpray source at 2000 V and 45˚C, and the transfer tube temperature was set to 305˚C. Mass spectrometry (MS) detection was performed with a resolution of 120,000 for full MS (320-1600 m/z scan range) and AGC target of 300,000. A full-MS1 scan (120,000 resolution, 60 ms accumulation time, AGC 3×10⁶) was followed by 20 data-dependent MS2 scans (60,000 resolution, 120 ms accumulation time, AGC 5×10⁵), with an isolation width of 1.6 m/z and normalized HCD energy of 25%.