Lcms 8060 triple quadrupole mass spectrometer

Liquid chromatography was performed on a Nexera XR 40 series HPLC (Shimadzu) using a Synergi 4 µM Fusion-RP 80 Å 150 × 2 mm column (Phenomenex). The column temperature was kept at 40 °C, and the sample tray was maintained at 4 °C. 10 μL samples were injected at a flow rate of 0.2 ml/min with 10 mM ammonium formate (pH 4.2) and methanol as mobile phases A and B, respectively. Metabolites were eluted using the following gradient: 0–8 min, 8–90% B; 8–10 min, 90% B; 10–10.1 min, 90–8% B; 10.1–20 min, 8% B. The LCMS-8060 triple quadrupole mass spectrometer with electro spray ionization (Shimadzu) was operated in the positive mode. Scheduled multiple reaction monitoring (MRM) was employed to monitor analyte parent ions to product ion formation. MRM conditions were optimized using authentic standard chemicals including NAM ([M + H]⁺ 123.00 > 80.00, 123.00 > 78.00, 123.00 > 53.00), NAD⁺ ([M + H]⁺ 664.00 > 136.00, 664.00 > 427.90, 664.00 > 523.95), cADPR ([M + H]⁺ 541.80 > 136.15, 541.80 > 427.90, 541.80 > 347.90), and ADPR ([M + H]⁺ 559.80 > 136.05, 559.80 > 427.95, 559.80 > 347.95). Data acquisition was performed using the LabSolutions LCMS v5.97 software, and data processing was carried out using LabSolutions Postrun (both Shimadzu). Metabolite products were quantified by scheduled MRM peak integration using calibration curves of standard chemicals.

All analyses were performed using the LCMS-8060 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with the ESI source operated in positive MRM ion mode following a previously validated method [8 (link)]. The chromatographic separation was performed using the UPLC Nexera X2 System (Shimadzu, Kyoto, Japan) equipped with the LC-30AD binary pump, DGU-20A5R degasser, CBM-20A controller, SIL-30AC autosampler, and CTO-20AC thermostated column oven. The separation of the derivatizated BAs was achieved using the Kinetex C8 (100 × 2.1 mm, 1.7 µm, Phenomenex, Torrance, CA, USA) column. The chromatographic separation conditions, MS/MS operation parameters (Table 2), and parameters of the monitored ion transitions (Supplementary Materials Table S1) are available in Supplementary Materials. Data acquisition and initial analysis were accomplished with LabSolutions 5.60 SP1 software. Further data processing and multivariate data analysis was performed using Orange v. 3.28 and Scikit-Learn v. 0.24 Python packages [21 ,22 ].

The assimilation ratio of newly added nitrogen sources at each time point was determined using stable isotope ¹⁵N-labelled Na¹⁵NO₃ or ¹⁵NH₄Cl. Synechocystis was transferred to the modified BG11 medium containing 5 mM Na¹⁵NO₃ or ¹⁵NH₄Cl 24 h after inoculation and cultivated under 1% (v/v) CO₂ and 100 μ mol photons m⁻² s⁻¹ at 30 °C. The culture medium corresponding to 5 mg of dry cell weight was recovered at 0, 4, and 24 h, and the intracellular metabolites were analyzed by CE-MS as described above or by LC-MS/MS MRM. The procedure of sample preparation was the same as described above Section 4.2 (measurement of the intracellular metabolite concentration) and LC-MS/MS MRM analysis was performed by employing Nexera X2 high-performance liquid chromatography system and a LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan), as described previously [28 (link)]. The ¹⁵N labeling rate was calculated as performed in ¹³C labeling experiments in previous reports [18 (link)]. The relative isotopomer abundance (m_i) for each metabolite in which the i¹⁵N atoms were incorporated is calculated as follows: $$m_i(\%)=\frac{M_i}{{{\displaystyle \sum }}_{j=0}^n{M}_j}\times 100$$
$${}_{}{}^{15}\mathrm{N} \mathrm{fraction}(\%)={\displaystyle \sum }_{i=1}^n\frac{i\times m_i}{n}$$
where M_i represents the isotopomer abundance of metabolite incorporating i¹⁵N atoms, and n is the number of nitrogen atoms in the metabolite. Statistical analysis was conducted using Welch’s t-test (* <0.05, ** <0.01).

The LC and MS measurement conditions were as follows: Serum samples were subjected to a Nexera UHPLC system and LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Co., Kyoto, Japan). An Acquity UPLC BEH C8 column (1.7 µm, 2.1 × 100 mm; Waters) was used with the following mobile phase compositions: 5 mM NH₄HCO₃/water (mobile phase A), acetonitrile (mobile phase B), and isopropanol (mobile phase C). The pump gradient was programmed as follows [time (%A/%B/%C)]: 0 min (95/5/0), to 8 min (70/30/0), 16 min (30/35/35), 28 min (6/47/47), 35 min (6/47/47), 35.1 min (95/5/0); it was then held for 38 min for equilibration. The flow rate was 0.35 mL/min, and the column temperature was 47 °C. The injection volume was 5 µL. SRM analysis was performed using positive/negative ion-switching mode ESI, with a collision energy of 46 eV. All data were analyzed using Microsoft Excel 2016.

In this work, we used our previously described^{14 (link)} LC-MS/MS system which consists of a Nexera X2 UHPLC interfaced with an LC-MS 8060 triple quadrupole mass spectrometer equipped with an electrospray ion source (Shimadzu, Kyoto, Japan). Chromatography was accomplished using an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm, Waters, Milford, USA) maintained at 40 °C during analysis.
Chromatographic analysis of CA, MCA and their stable isotope labeled internal standards was achieved using 2% methanol containing 0.5 g/L PFOA for 4 min as mobile phase. The methanol concentration was increased to 85% at 4 min and to 95% at 6 min using linear gradient. A 2 min column re-conditioning step with 2% methanol containing 0.5 g/L PFOA was included after each injection. A constant flow rate of 400 μL/min was used in all experiments.
Analysis by MS/MS was in the positive ion mode using argon as collision gas with collision energy of 22 eV, capillary voltage of 3.4 kV and cone voltage of 35 V. Desolvation temperature of 120 °C and ion source temperature of 350 °C were applied. Detection was in the multiple reaction monitoring mode with m/z of 485 > 151 and 489 > 151 for CA and d4-CA, respectively. MCA and d3-MCA were detected using m/z 499 > 151 and 502 > 151 as previously described^{12 ,14 (link)}.