Monitoring Membrane Lipidome Turnover by Metabolic ¹⁵N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic ¹⁵N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 10⁶ (<i>m</i>/<i>z</i> 200) baseline resolved peaks of ¹³C isotopes of unlabeled and monoisotopic peaks of ¹⁵N labeled lipids (Δ<i>m</i> = 0.0063 Da). Therefore, the rate of metabolic ¹⁵N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells