Q exactive instrument

The plasma TMAO concentrations were quantified using liquid chromatography-mass spectrometry that employed a Q Exactive instrument (Thermo Scientific, San Jose, CA) equipped with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific), and separation was carried out at 30 °C in an Xbridge amide column (4.6 × 150 mm, 3.5 mm, Waters, Milford, MA). The mobile phase consisted of a combination of 5% acetonitrile containing 5 mM ammonium acetate in MilliQ water (solution A) and 5 mM ammonium acetate in acetonitrile (solution B). The chromatogram was run under isocratic conditions at a flow rate of 0.7 mL/min, as follows: A/B = 95/5. A heated electrospray ionization (HESI) ion source was used for the ionization. The HESI parameters were optimized as follows: sheath gas flow rate 53 units, auxiliary gas unit flow rate 14, capillary temperature 269 °C, auxiliary gas heater temperature 45 °C, spray voltage 2500 V, and S lens RF level 55.

Four mature leaves at the mid-bottom position were collected from each plant and subjected to air curing for 7 weeks. The concentrations of amino acids and nicotine in the cured leaf lamina were determined by UHPLC-MS (Dionex Ultimate 3000). Samples of ground tobacco (~30 mg) were extracted with ethanol–water (1:1; 6 mL) for 45 min at 50 °C. The centrifuged extracts were diluted 10-fold. Liquid chromatographic separation was performed on an amide column (Waters Acquity BEH amide, 2.1- x 150-mm; 1.7 µm) at 45 °C; elution was performed with a gradient of 2 mM ammonium formate in water + 0.25% formic acid (eluent A) and acetonitrile + 0.1% formic acid (eluent B) by applying an elution gradient (0 min – 10% A, 0.5 min – 10% A, 4 min – 60% A, 4.5 min – 60% A, 4.6 min – 10% A, 6.8 min – 10% A; flow rate, 0.5 mL/min). For mass spectrometric detection of amino acids, a Q Exactive instrument (Thermo Scientific, Basel, Switzerland) was used in positive-ion electrospray ionization (ESI) mode for acquiring full-scan mass spectra. Nicotine concentrations in the extracts were calculated by using the peak-area integration method (peak in the 260-nm trace of a photodiode UV-VIS detector).

Peptides were separated with an online 3000 RSLCnano system. Samples were trapped on an Acclaim PepMap nanotrap column (C18, 3 μm, 100 Å, 75 μm × 20 mm), and separated on an Acclaim PepMap RSLC column (C18, 2 μm, 100 Å, 75 μm × 50 cm; Thermo scientific). Next, HiRIEF-fractionated peptides were separated on a gradient of A (5% DMSO, 0.1% Formic acid; FA) combined with B (90% Acetonitrile; ACN, 5% DMSO, 0.1% FA), where B ranged from 3% to 37%. Samples were run for 50 min at a flowrate of 0.25 μL/min. The Q Exactive instrument (Thermo Fischer Scientific, San Jose, CA, USA) was operated in a data-dependent manner, where the top 5 precursors were selected for HCD fragmentation and MS/MS. The survey scan was performed at 70,000 resolution over a range of 300–1600 m/z, with a maximum injection time of 100 ms and target of 1 × 10⁶ ions. HCD fragmentation spectra were generated with a maximum ion injection time of 150 ms and an AGC of 1 × 10⁵. Then, fragmentation was performed at 30% normalized collision energy, with 35,000 resolution. Precursors were isolated with a width of 2 m/z and placed on the exclusion list for 70 s. For 4-h gradients, we used a top 10 method, with a survey scan over the range of 400–1600 m/z and a maximum injection of 140 ms. Single and unassigned charge states were rejected from precursor selection.

For each sample, peptides were loaded onto a 2cm C18 trap column (cat. no.164705, Thermo Fisher), connected in-line to a 75 cm C18 reverse-phase analytical column (cat. no. ES805, Thermo EasySpray) using 100% Buffer A (0.1% Formic acid in water) at 750bar, using the Thermo EasyLC 1000 HPLC system, and the column oven operating at 45°C. Peptides were eluted over a 200-minute gradient ranging from 6 to 60% of 80% acetonitrile, 0.1% formic acid at 250 nL/minute, and the Q-Exactive instrument (Thermo Fisher Scientific) was run in a DD-MS2 top10 method. Full MS spectra were collected at a resolution of 70,000, with an AGC target of 3×10⁶ or maximum injection time of 20 milliseconds and a scan range of 300–1750 m/z. The MS2 spectra were obtained at a resolution of 17,500, with an AGC target value of 1×10⁶ or maximum injection time of 60 milliseconds, a normalized collision energy of 25 and an intensity threshold of 1.7 ×10⁴. Dynamic exclusion was set to 60 seconds, and ions with a charge state < 2 or unknown were excluded. MS performance was verified for consistency by running complex cell lysate quality control standards, and chromatography was monitored to check for reproducibility.

Peptides from biological triplicates of each culture condition were loaded on the mass spectrometer by reverse phase chromatography through an inline 50 cm C18 column (Thermo EasySpray ES803) connected to a 2 cm long C18 trap column (Thermo Fisher 164705) using a Thermo EasyLc 1000 HPLC system. Peptides were eluted with a gradient of 4.8–48% (v/v) ACN, 0.1% (w/v) FA at 250 nL min⁻¹ over 260 min (samples from single strain cultures) or 140 min (SCX fractionated samples from co cultures) and analysed on a Q-Exactive instrument (Thermo Fisher Scientific) run in a data-dependent manner using a Top 10 method. Full MS spectra were collected at 70,000 resolution, with an AGC target set to 3 × 10⁶ ions or maximum injection time of 20 ms. Peptides were fragmented via higher-energy collision dissociation (normalized collision energy = 25). The intensity threshold was set to 1.7 × 10⁶, dynamic exclusion to 60 s and ions with a charge state <2 or unknown species were excluded. MS/MS spectra were acquired at a resolution of 17,500, with an AGC target value of 1 × 10⁶ ions or a maximum injection time of 60 ms. The scan range was limited from 300–1750 m/z.

For lipid quantification by shotgun MS, 700 μL of a mixture of internal standards in MTBE/MeOH (5:1.5; [vol/vol]) were added to the dried lipid fractions. MS analyses were performed as previously described (74 (link)) on a Q Exactive instrument (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source TriVersa NanoMate (Advion BioSciences) using nanoelectrospray chips with the diameter of spraying nozzles of 4.1 μm. The ion source was controlled by the Chipsoft 8.3.1 software (Advion BioSciences). Spectra was filtered based on repetition rate as previously described and analyzed by a laboratory-developed script (75 (link), 76 (link)). Lipids were identified by LipidXplorer software (77 (link)).