Ultimate 3000 uplc

Mass spectrometry was carried out on a nanoflow Ultimate 3,000 UPLC (Thermo Scientific, San Jose, CA) coupled to Impact II mass spectrometer with a CaptiveSpray source equipped with a nanoBooster device (Bruker Daltonik, Bremen, Germany) operated at 1,800 V. For each fractionated sample, 1 μl of the sample was loaded on a C18 PepMap100 nano-Trap column (300 μm ID × 5 mm, 5 micron 100 Å) at a flow rate of 3,000 nl/min. The trap column was then switched in line with the analytical column ProntoSIL C18AQ (100 μm ID × 150 mm 3 micron 200 Å) (nanoLCMS Solutions, Gold River, CA). The reverse-phase elution gradient was from 2 to 20% to 45% solvent B over 60 min, total 88 min at a flow rate of 600 nl/min. Solvent A was LCMS-grade water with 0.1% formic acid; solvent B was LCMS-grade acetonitrile with 0.1% formic acid.
The fractionated samples were measured in data-dependent MS/MS mode, where the acquisition speed was 2 Hz in MS and 2–32 Hz in MS/MS mode depending on precursor intensity. Ten precursors were selected in the m/z 150–2,200 range, with one to eight charged peptides selected. The analysis was performed in positive ionization mode with a dynamic exclusion of 60 s.

For extraction of B[α]P, the samples were dissolved in a sodium acetate buffer (0.2 M, pH 4). The samples were mixed with acetone and then extracted with ethyl acetate three times. The ethyl acetate extract was dehydrated with anhydrous sodium sulfate, as well as evaporated by a nitrogen evaporator (Organomation, Berlin, MA, USA) at a temperature below 40 °C. After that, the dry residue was dissolved in hexane before solid phase extraction through a Sep-Pak^® Vac 6 cc (1 g) C18 cartridges (Waters, Milford, MA, USA) with DCM and hexane. The organic solvents were completely evaporated under nitrogen gas at a temperature below 40 °C. For the analysis, dry residue samples were dissolved in ACN and filtered with a PVDF 0.2 μm syringe filter. The chromatography analysis was conducted using an Ultimate 3000 UPLC (Thermo Fisher Scientific, Waltham, MA, USA) with a reversed phase column (Zorbax Eclipse XDB C18 column 4.6 × 250 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). The mobile phase was ACN (mobile phase A) and water (mobile phase B). The gradient elution was 80% A + 20% B at 0–20 min, 100% A at 20–25 min, and 80% A + 20% B at 25–30 min with a flow rate of 1.0 mL/minute. The injection volume and UV wavelength was 20 μL and 280 nm, respectively.

High-resolution LC-MS/MS analysis was carried out on an Ultimate 3000 UPLC (Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific). Mass accuracy was determined to be better than 1 ppm. Liquid chromatography was performed using Phenomenex Kinetex C₁₈, 2.6 μm, 100 × 2.1 mm at a flow rate of 400 μl·min⁻¹ over 25 min. Mobile phases A and B were identical to the method above but with a gradient for B of 15–28% over 0–1 min, 28–42% over 1–4 min, 42–60% over 4–6 min, 60–80% over 6–16 min, 80–95% over 16–19 min, and held at 95% over 19–22 min followed by 3-min re-equilibration to 15%. Injection volume was 10 μl. Electrospray ionization was used in negative mode with a sheath gas flow rate of 60 units and capillary temperature at 320 °C. Data-dependent MS/MS of m/z 351 was carried out with a resolving power of 35,000.

Untargeted metabolic profiling was performed on an ultrahigh performance liquid chromatography-high resolution mass spectrometry (Model: Thermo Ultimate 3000 UPLC and Thermo QExactive mass spectrometer) platform at the University of Florida Southeast Center for Integrated Metabolomics (SECIM). All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 70,000 at m/z 200 as separate injections. Chromatographic separation was attained on an ACE Excel 2 C18 PFP100 × 2.1 mm, a 2 μm particle size column with mobile phase A as 0.1% formic acid in water, and mobile phase B as acetonitrile, at a flow rate of 350 μL/min with a run time of 16.8 min, mass resolution of 35,000 at m/z 200, and mass range of 70–1000 m/z. Injection volume was 4 μL for negative ion mode and 2 μL for positive ion mode. The total run time per sample was 20.5 minutes. Probe (HESI probe) temperature was maintained at 350°C for both positive and negative run with a spray voltage of 3500 V and a capillary temperature of 320 °C [42 (link)].

c407 was quantified by an UPLC-MS/MS system, an Ultimate 3000 UPLC and a TSQ Vantage MS/MS detector (Thermo Fischer, Waltham, Masssachusetts, USA). Briefly, after adding 50 µl of the deuterated isotope (100 ng/ml) to 100 µL of plasma, 500 µL of acetonitrile was added for protein precipitation. Samples were then vortex-mixed for 30 s, and subsequently centrifuged at 20,000g for 10 min. The supernatant was then evaporated to dryness under a stream of nitrogen and the dry residue was reconstituted with 200 µl of mobile phase (ammonium acetate 10 mM: methanol; 80:20). Ten µL were then injected to the UPLC-MS/MS system. Chromatographic separation was performed with a Kinetex C-18, 100 × 2.1 mm column (Phenomenex, Le Pecq, France) and the mobile phase flow was set at 0.4 mL/min. The monitored m/z transitions were 311 to 169 for c407 and 315 to 172 for its deuterated isotope. Spray voltage, vaporizer temperature and capillary temperature were 4000 V, 350 °C and 300 °C respectively. The calibration range was 1–10 000 ng/mL.

Proteomic analysis was performed on an ultra-high-resolution Orbitrap Fusion mass spectrometer equipped with a nanoflow Ultimate 3000 UPLC (both from Thermo Fisher, Germany) as described in Ref.^{91 (link)}. A survey of metabolomic compounds was conducted on a high-resolution G6550 Q-TOF mass spectrometer with a 1290 Infinity UPLC (both from Agilent, Germany). Details are given in Supplementary Appendix B (sections no. 3 and no. 7 for proteomic analysis, and sections no. 5 and no. 8 for metabolomic analysis). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE^{93 (link)} partner repository with the dataset identifier PXD035863 and 10.6019/PXD035863.