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Originally published In Press as doi:10.1194/jlr.D500007-JLR200 on April 16, 2005

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Journal of Lipid Research, Vol. 46, 1548-1560, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization

Xianlin Han1,*,{dagger}, Kui Yang*, Hua Cheng*, Kora N. Fikes* and Richard W. Gross*,{dagger},§,**

* Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, St. Louis, MO 63110
{dagger} Departments of Medicine, Washington University School of Medicine, St. Louis, MO 63110
§ Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110
** Chemistry, Washington University School of Medicine, St. Louis, MO 63110

Published, JLR Papers in Press, April 16, 2005. DOI 10.1194/jlr.D500007-JLR200

1 To whom correspondence should be addressed. e-mail: xianlin{at}wustl.edu

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions.

Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.

Abbreviations: 2D, two-dimensional; DMAP, dimethylaminopyridine; ESI, electrospray ionization; Fmoc-Cl, fluorenylmethoxylcarbonyl chloride; m:n, acyl chain containing m carbons and n double bonds; MS, mass spectrometry; NAC, 2-(2-naphthyl)acetyl chloride; NL, neutral loss; PE, ethanolamine glycerophospholipid; PI, precursor ion; PlsEtn, plasmenylethanolamine; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol

Supplementary key words electrospray ionization-mass spectrometry • fluorenylmethoxylcarbonyl derivatization • lipidome • lipidomics • lysophosphatidylethanolamine • multidimensional mass spectrometry • plasmalogen


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