Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR

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69 Scopus citations

Abstract

We have coupled 2D-NMR and infusion FT-ICR-MS with computer-assisted assignment to profile 13C-isotopologues of glycerophospholipids (GPL) directly in crude cell extracts, resulting in very high information throughput of >3000 isobaric molecules in a few minutes. A mass accuracy of better than 1 ppm combined with a resolution of 100,000 at the measured m/z was required to distinguish isotopomers from other GPL structures. Isotopologue analysis of GPLs extracted from LCC2 breast cancer cells grown on [U-13C]-glucose provided a rich trove of information about the biosynthesis and turnover of the GPLs. The isotopologue intensity ratios from the FT-ICR-MS were accurate to ≈1% or better based on natural abundance background, and depended on the signal-to-nose ratio. The time course of incorporation of 13C from [U-13C]-glucose into a particular phosphatidylcholine was analyzed in detail, to provide a quantitative measure of the sizes of glycerol, acetyl CoA and total GPL pools in growing LCC2 cells. Independent and complementary analysis of the positional 13C enrichment in the glycerol and fatty acyl chains obtained from high resolution 2D NMR was used to verify key aspects of the model. This technology enables simple and rapid sample preparation, has rapid analysis, and is generally applicable to unfractionated GPLs of almost any head group, and to mixtures of other classes of metabolites.

Original languageEnglish
Pages (from-to)201-208
Number of pages8
JournalAnalytica Chimica Acta
Volume651
Issue number2
DOIs
StatePublished - Oct 5 2009

Bibliographical note

Funding Information:
This work was supported in part by National Science Foundation EPSCoR grant # EPS-0447479 , NIH Grant Number P20RR018733 from the National Center for Research Resources , 1R01CA118434-01A2 from the National Cancer Institute , the Kentucky Challenge for Excellence , Susan G. Komen Foundation BCTR0503648 , and the Brown Foundation .

Keywords

  • Fourier transform-ion cyclotron resonance-mass spectrometer
  • Nuclear magnetic resonance
  • Phospholipid biosynthesis
  • Stable isotopomer analysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

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