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

Abstract

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic N 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 × 10 (m/z 200) baseline resolved peaks of C isotopes of unlabeled and monoisotopic peaks ofN labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic N 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.

Restricted spectral accuracy analysis to identify the single correct organic compound elemental‐composition from Orbitrap accurate mass data lists obtained at very high resolution

“…Some recent work further delineating coalescence has appeared but is largely an extension of Gorshkov's experimental design, purposely creating mixtures that produce closely spaced isotope lines . Workers in the field of metabonomics and lipidomics have also made recent contributions toward understanding more about coalescence in their particular contexts . The focus of our work is avoiding coalescence.…”

Sterols as dietary markers forDrosophila melanogaster

“…PRM spectra were acquired from 4 min to 10 min. For PRM micro scans were set to 1, isolation window to 0.8 Da, normalized collision energy to 12.5%, AGC to 5 × 10 4 and maximum injection time to 3000 ms. All spectra were pre-processed using repetition rate filtering software PeakStrainer [26] and stitched together by an in-house developed script [17]. Lipids were identified by LipidXplorer software [27].…”

Lipidomics from sample preparation to data analysis: a primer

“…For more information on the correlation between mass resolution and identification ambiguities, readers are referred to Bielow et al, who cover this topic in depth [32]. Instrumentation with a mass resolution beyond 500,000 makes it possible to perform isotopic labeling experiments and subsequent metabolic flux analysis with isotopes that have naturally low abundances, such as the technique proposed by Schuhmann et al for 15 N labeling in human HepG2 cells [33]. A recently published and interesting approach to addressing the ion suppression challenges arising from direct infusion is spectral stitching [33,34].…”

“…Instrumentation with a mass resolution beyond 500,000 makes it possible to perform isotopic labeling experiments and subsequent metabolic flux analysis with isotopes that have naturally low abundances, such as the technique proposed by Schuhmann et al for 15 N labeling in human HepG2 cells [33]. A recently published and interesting approach to addressing the ion suppression challenges arising from direct infusion is spectral stitching [33,34]. The proposed acquisition protocol parses the mass range of the fullscan spectrum into smaller selected ion monitoring (SIM) mass ranges of between 20 and 50 Da, which are acquired sequentially and then 'stitched' together by the software into one large full-scan spectrum.…”