Kinetex c8 column

The ACQUITY UPLC system (Waters, Milford, MA, USA) consisted of a binary solvent manager, a vacuum degasser, a column heater and sample manager. The column temperature was maintained at 50°C. The samples were injected onto a Kinetex C8 column, 50 × 2.1 mm, 2.6 μm particle diameter (Phenomenex, Torrance, CA, USA). The acylcarnitines were separated by a linear gradient between solution A (0.1% formic acid in H₂O) and solution B (0.1% formic acid in methanol). The gradient was as follows: at T = 0 min: 36% A, 64% B, flow 0.4 mL/min towards T = 6 min: 0% A, 100% B, flow 0.4 mL/min; T = 6–11 min: 0% A, 100% B, flow 0.4 mL/min, and T = 11–11.1 back to 36% A, 64% B, flow 0.4 mL/min. A Quattro Premier XE (Waters, Milford, MA, USA) was used in the positive electrospray ionization mode. Nitrogen was used as desolvation gas (900 L/h) and cone gas (50 L/h). Desolvation temperature was 350°C, capillary voltage was 3.5 kV and the source temperature was 130°C. Argon was used as collision gas (2.5 x 10e-3 mbar). For the very long-chain acylcarnitines and lysophosphatidylcholines (lysoPC) multiple reaction monitoring (MRM) traces were acquired with optimized cone voltage and collision energy for each transition with a dwell time of 0.01 s (Table 1).

LC/ESI-MS was performed using the high-pressure LC installed LCMS-8060 (Shimadzu Corporation) system. The LC/MS/MS Method Package for Phospholipid Profiling (Shimadzu Corporation) was used according to the manufacturer's instructions to analyze the PL. Kinetex C8 column (Kinetex C8, 150 mm × 2.1 mm i.d., 3.6 μm particle size; Phenomenex, Torrance, CA, USA) with mobile phases A (20 mM ammonium formate in water) and B (acetonitrile-isopropanol 1:1 v/v) were employed for LC separation. The mobile phase B gradient was programmed as 20% (0 min)-20% (1 min)-40% (2 min)-92.5% (25 min). The column oven temperature was 45 °C. To analyze the PM, LC/MS/MS Method Package for Primary Metabolites, Ver. 2, was used. Discovery HS F5-3 column (150 mm × 2.1 mm I.D. ×, 3 µm particle size; Merk & Co., Kenilworth, NJ, USA) with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) was used for LC separation. Furthermore, mobile phase B gradient was programmed as 0% (0 min)-25% (51 min)-35% (11 min)-95% (20 min). The column oven temperature changed to 40 °C.

The purity of the biosynthesized proteins and chemically synthesized peptides was assessed by LC-MS (Agilent 1260 Infinity-6120 quadrupole mass spectrometer). All preparations were analyzed by reverse-phase Kinetex C8 column (2.6 μm, 100 Å, LC Column 75 × 2.1 mm; Phenomenex) with a linear gradient of 10–80% ACN over 10 min at a flow rate of 1.0 ml/min using aqueous 0.05% TFA and aqueous 0.05% TFA/90% ACN elution buffers. All proteins and FGF21 peptides were obtained to >90% purity. The purity within the set of 19C26 Ala-scan peptides was assessed by their 214 nm absorbance LC profile, and the concentrations were appropriately adjusted for the in vitro assays to reflect the concentration of the desired peptide in the preparation. The concentration of each protein and peptide was determined based on their UV absorbance on NanoDrop spectrophotometer (ThermoFisher Scientific), and the extinction coefficients for respective sequences were obtained with online tools (Prot pi peptide or ExPASy ProtParam). FGF21, FGF19, FGF21-19A protein preparations used for in vivo studies were purified from endotoxin by ToxinEraser Endotoxin removal kit (GenScript) to ensure 0.5 EU/mg or less endotoxin.