Q exactive plus quadrupole orbitrap mass spectrometer

Mass spectrometry was performed on a Q-Exactive Plus™ quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, United States) in negative ion mode. The scan mass range was set at m/z 100–1,200. The parameter settings were as follows: a full scan and fragment spectral resolution of 70,000 FWHM and 17, 50 FWHM, respectively; the capillary temperature was 350°C; auxiliary gas heater temperature was 350°C; spray voltage was −3.2 KV; sheath gas flow rate was 40 Arb; auxiliary gas flow rate was 15 Arb; S-lens RF level was set at 50. The acquisition mode of stepped NCE (normalized collision energy) was used with settings of 30, 50, and 70 eV. The accumulated resultant fragment ions were injected into the Orbitrap mass analyzer for single-scan detection.
Considering the possible elemental composition of the RM components, the types and quantities of expected atoms were set as follows: carbon ≤50, hydrogen ≤200, oxygen ≤20, nitrogen ≤3. The accuracy error threshold was fixed at 5 ppm.

Melting points (Mps) were determined with a Kofler hot-stage apparatus and were uncorrected. Elemental analyses were performed with a Perkin-Elmer CHN analyzer model. Thin-layer chromatography was performed on silica gel 60 F254 (layer thickness 0.2 mm, Merck, Rahway, NJ, USA); eluents: (A) 70% ethyl acetate/30% hexane, (B) 30% ethyl acetate/70% hexane. The spots were detected with I₂ or UV (365 nm) after spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid and heating at 100–120 °C for 10 min. Flash chromatography was performed on silica gel 60, 40–63 μm (Merck). ¹H NMR spectra were recorded in DMSO-d₆ or CDCl₃ solution with a Bruker DRX-500 instrument at 500 MHz. ¹³C NMR spectra were recorded with the same instrument at 125 MHz under the same conditions. Mass spectrometry: Full-scan mass spectra of the newly synthesized compounds were acquired in the range of 100 to 1100 m/z with a Q Exactive Plus quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray (HESI). Analyses were performed in positive ion mode using flow-injection mass spectrometry with a mobile phase of 50% aqueous acetonitrile containing 0.1 v/v% formic acid (0.3 mL/min flow rate). Aliquots of 5 µL of samples were injected into the flow. The ESI capillary was adjusted to 3.5 kV, and N₂ was used as a nebulizer gas.

An online nanoflow liquid chromatography-tandem mass spectrometry was used to assess the labeled peptides via the 9RKFSG2_NCS-3500R system (Thermo, Fort Pierce, FL, USA) connected to a Q Exactive Plus quadrupole orbitrap mass spectrometer (Thermo, Fort Pierce, FL, USA) through a nanoelectrospray ion source [32 (link)]. Briefly, the C18-reversed-phase column (75 μm × 25 cm, Thermo, Fort Pierce, FL, USA) was equilibrated with solvent A (A:2% ACN with 0.1% formic acid) and solvent B (B: 80% ACN with 0.1% formic acid). The peptides were eluted as follows: 0–63 min, 5–23% B; 63–82 min, 23−29% B; 82–90 min, 29−38% B; 90−92 min, 38−48% B; 92–94 min, 48–100% B; 94–120 min, 100–0% B at a flow rate of 300 nL/min.
The Q Exactive Plus was operated in the data-dependent acquisition mode (DDA) to switch between full-scan MS and MS/MS acquisition automatically. The orbitrap was used to obtain full-scan MS spectra (m/z 350–1500) with a 60,000 resolution. The automatic gain control (AGC) target was 3e6, and the maximum fill time was 20 ms. The top 20 most intense precursor ions were selected as collision cells for fragmentation using higher-energy collision dissociation (HCD). The MS/MS resolution automatic gain control (AGC) target, maximum fill time, and dynamic exclusion were 15000 (at m/z 100), 5e4, 45 ms, 45 ms, and 20 s, respectively.

Untargeted lipidomics was performed at the Harvard Center for Mass Spectrometry following a published protocol (Zhang et al., 2019 (link)). Data were acquired in MS/DD–MS² (top5) mode on a Q Exactive Plus quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) online with an Ultimate 3000 HPLC (Thermo Fisher Scientific). Positive and negative ionization modes were acquired separately. Raw files (.raw) were converted into .mzXML via msconvert (Chambers et al., 2012 (link)) and analyzed in R using the XCMS package (Smith et al., 2006 (link)). The identified lipid species were verified manually in using Xcalibur (Thermo Fisher Scientific), taking into account MS¹ and MS² data.

Hepatocytes were extracted with –20°C isopropanol (100 μL per ~10⁶ cells), vortexed, and centrifuged at 16,000g for 10 minutes at 4°C. The supernatant was collected for liquid chromatography–mass spectrometry (LC-MS) analysis. A Q-Exactive Plus Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) operating in positive ion mode was coupled via electrospray ionization and used to scan from m/z 290 to 1,200 at 1 Hz and 140,000 resolution. LC separation was done on an Atlantis T3 Column (2.1 mm × 150 mm, 3 μm particle size, 100 Å pore size; Waters) using a gradient of solvent A (1 mM ammonium acetate, 35 mM acetic acid in 90:10 water/methanol) and solvent B (1 mM ammonium acetate, 35 mM acetic acid in 98:2 isopropanol/methanol). The flow rate was 150 μL/min. The LC gradient was as follows: 0 minutes, 25% B; 2 minutes, 25% B; 5.5 minutes, 65% B; 12.5 minutes, 100% B; 16.5 minutes, 100% B; 17 minutes, 25% B; 30 minutes, 25% B. The autosampler temperature was 4°C, and the injection volume was 3 μL. Data were analyzed using the Compound Discoverer (Thermo Fisher Scientific) and Maven software.