Infinitylab poroshell 120 hilic z column

The total osmolyte content was quantified by liquid chromatography mass spectrometry (LC-MS) on extracted samples using an Agilent 6540 UHD Accurate Mass QToF LCMS with 1290 HPLC System. Sample injection volumes were 1 µL, auto-loaded and run on an Agilent InfinityLab Poroshell 120 HILIC-Z column, (2.1 × 100 mm) (1.9 µm × 120 Å pore size) with a pre-programmed ID tag. Separation solvents were solvent A (20 mM NH₄HCO₂ in 100% ddH₂O, pH adjusted to 3.0 with HCOOH) and solvent B (20 mM NH₄HCO₂ in ddH₂O and 1:10 acetonitrile solution, pH adjusted to 3.0 with HCOOH). Electrospray ionisation mass spectrometry (ESI-MS) source settings are listed in Table 2. Aqueous external standards of betaine, proline, ectoine and hydroxyectoine (Sigma-Aldrich), dissolved in ddH₂O and serial diluted to 0.36 µg mL⁻¹ were used to calibrate the data. Acquired data were processed by MassHunter Qualitative Analysis B.05.00. Results were calculated using ratios of the peak area of the analytes to their standards. Results from the analysis were standardised and recorded as a response factor via the following equation.
Response factor (RF) = ((PA_s/PA_x)/T_c) × 10⁹ whereby PA_s is the peak area of sample, PA_x is peak area of known standard signal and T_c is the total cell count of the sample. Significance of differences between the samples were determined using two-sample t-tests.

4-MBQ was mixed with CML or amino acids at equimolar concentrations, and then the solution was diluted to 25 μM with pH 7.4 PBS buffer and allowed to react at 25 °C for 1 h. The reaction product solutions were filtered through a 0.22 μM syringe filter. Samples (2 μL) were injected into a UPLC system (AB Sciex Inc., Framingham, MA, USA) equipped with an Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm) and eluted at a flow rate of 0.3 mL/min with eluent A consisting of 0.1% ammonium formic acid and eluent B consisting of methanol. Gradient elution was performed as follows: 0 min, 5% B; 4 min, 5% B; 8 min, 50% B; 10 min, 95% B; 12 min, 95% B; 13 min, 5% B; and 15 min, 5% B. The column was operated at 30 °C. Other mass spectrometry conditions are provided in Section 2.3.

To determine nucleotides, cell extracts were analysed using an UHPLC system coupled to a 6490 QqQ mass spectrometer (Agilent Technologies). Cell extracts were injected (5 µl) and metabolites were separated using an InfinityLab Poroshell 120 HILIC-Z column (2.7 µm, 2.1 × 100 mm, Agilent). The mobile phases used for the metabolite separation were A: 50 mM ammonium acetate with 5 µM medrionic acid; and B: acetonitrile. The chromatographic gradient was isocratic for 0.5 min at 80% B, from 0.5 to 7.5 min decreased to 70% B and from 7.5 to 8.5 min decreased to 50%, and maintained for 30 sec. From 9.0 min to 9.2 min the percentage of B rose quickly to 80% and finally the column was equilibrated at 80% B until min 11. Flow rate was 0.7 mL/min. The QqQ mass spectrometer worked in MRM mode using the transitions in Supplementary Table S3 to determine nucleotides. The electrospray ionization source (ESI) worked in positive and negative mode. The quantification of nucleotides was based on peak areas; the indicated relative concentrations correspond to the peak area/cell number (Supplementary Dataset).

This study focused on several metabolites involved in Glycolysis, TCA cycles, pentose phosphate pathway, urea cycles and several key amino acids in PRODH/POX-dependent pathways. Testing metabolites are summarized in Table S2 (Supplementary Materials). All stock solution of reference metabolites were prepared in acetonitrile to obtain 1000 ppm (mg·mL⁻¹). LC-MS/MS analysis was performed using an Agilent 1200 LC coupled to an Agilent 6470 Triple quadrupole (Agilent Technologies, Santa Clara, CA, US) with an InfinityLab Poroshell 120 HILIC-Z column (Agilent Technologies, Santa Clara, CA, US) for (hydrophilic liquid chromatography (HILIC) interaction. The platform was operated in a multiple reaction monitoring (MRM) in negative mode using an electrospray ionization (ESI) source. The optimized transition of amino acid metabolites is listed in Table S3 (Supplementary Materials). The injection volume was 2 µL. Mobile phase A was 10 mM ammonium acetate adjusted to pH = 9 with ammonia, with 2.5 mM InfinityLab deactivator additive (Agilent, P-N. 5191-4506). Mobile phase B was 10 mM ammonium acetate adjusted to pH = 9 in H₂O/ACN (15:85, v/v) with 2.5 mM of the same deactivator. The flow was constant at 0.250 mL/min. The chromatographic gradient is described in reference [29 ].

For metabolic flux experiments, ¹³C-palmitate tracing was conducted using LC/MS at LipidALL Technologies. Cells (1 × 10⁷) were incubated for 8 h at 37 °C in RPMI-1640 medium with 10% FBS. For ¹³C-palmitate incorporation in metabolites, cells were incubated in growth medium with 50 μM [U-¹³C] palmitate for 24 h. Then, cells were quickly washed with 1×PBS and fixed with pre-cooled methanol (HPLC-MS grade, Millipore) for 30 min at −80 °C. The extraction method was referenced to a previous paper, but with modification [52 (link)]. Samples were incubated for 30 min at 1500 rpm and 4 °C and then centrifuged for 10 min at 12,000 rpm and 4 °C. The supernatant fractions were placed into clean 1.5-ml centrifuge tubes and dried using a SpeedVac. The dried extracts were dissolved with 50% acetonitrile in water and the upper layer liquids were collected for LC–MS analysis. The InfinityLab Poroshell 120 HILIC-Z column (2.1 mm × 50 mm, 2.7 μm, Agilent Technologies, Germany) was used in this study. Ultra-performance Liquid Chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to the Quadrupole-TOF MS 5600 Triple TOF Plus, AB SCIEX, Singapore) was used to acquire metabolome data.