Viva c4 column

Oligonucleotides were obtained from Integrated DNA Technologies. Enzymes are reagents used for cloning were obtained from New England BioLabs Inc. Non-canonical amino acids were purchased from Chem-Impex. DNA sequencing was performed by Genewiz. Protein LC-MS analysis was performed on an Agilent 6520 Accurate Mass QToF LC-MS LC-MS ESI positive in high resolution mode. The LC-MS was equipped with a Restek Viva C4 column. All other chemical reagents and solvents were obtained from chemical suppliers (Acros, Fisher Scientific, or Sigma-Aldrich) and used without further purification.

Negative ion electrospray ionization (ESI) LC-MS analysis of extracted CL was conducted on a Thermo Scientific (San Jose, CA) Vantage TSQ mass spectrometer with Thermo Accela UPLC operated by Xcalibur software. Separation of lipid was achieved by a Restek 150 × 2.1 mm (5 µm particle size) Viva C4 column at a flow rate of 260 µL/min. The mobile phase contained 10 mM ammonium formate in solvent A: acetonitrile:water (60:40, v:v); solvent B: 2-propanol:acetonitrile (90:10, v:v); and a gradient elution in the following manner was applied: 68% A, 0–1.5 min; 68−55% A, 1.5–4 min; 55−48% A, 4–5 min; 48−42% A, 5–8 min; 42−34% A, 8–11 min; 34−30% A, 11–14 min; 30−25% A, 14–18 min; 25−3% A, 18–23 min; 3−0% A, 25–30 min and kept at 0% A for 5 min. The tetramyristoyl CL internal standard (m/z 1240, [M – H]⁻) was eluted at 13.6 min, and the tetralinoleoyl CL (m/z 1448, [M – H]⁻) eluted at 14.4 min. Calculation of tetralinoleoyl CL content was based on the ratio of peak area of (18:2/18:2)₂-CL and (14:0/14:0)₂-CL from lipid extracts of cells incubated with PBS and CL-ND.