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LILY-lipidome isotope labeling of yeast: in vivo synthesis of 13C labeled reference lipids for quantification by mass spectrometry

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Abstract

Quantification is an essential task in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity and their dynamic range of the lipidome. In this work, we introduce lipidome isotope labeling of yeast (LILY) in order to produce (non-radioactive) isotopically labeled eukaryotic lipid standards in yeast for normalization and quantification in mass spectrometric assays. More specifically, LILY is a fast and efficient in vivo labeling strategy in Pichia pastoris for the production of 13C labeled lipid library further paving the way to comprehensive compound-specific internal standardization in quantitative mass spectrometry based assays. More than 200 lipid species (from PA, PC, PE, PG, PI, PS, LysoGP, CL, DAG, TAG, DMPE, Cer, HexCer, IPC, MIPC) were obtained from yeast extracts with an excellent 13C enrichment >99.5%, as determined by complementary high resolution mass spectrometry based shotgun and high resolution LC-MS/MS analysis. In a first proof of principle study we tested the relative and absolute quantification capabilities of the 13C enriched lipids obtained by LILY using a parallel reaction monitoring based LC-MS approach. In relative quantification it could be shown that compound specific internal standardization was essential for the accuracy extending the linear dynamic range to four orders of magnitude. Excellent analytical figures of merit were observed for absolute quantification for a selected panel of 5 investigated glycerophospholipids (e.g. LOQs around 5 fmol absolute; typical concentrations ranging between 1 to 10 nmol per 108 yeast cell starting material; RSDs <10% (N = 4)).

Graphical abstract: LILY-lipidome isotope labeling of yeast: in vivo synthesis of 13C labeled reference lipids for quantification by mass spectrometry

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Publication details

The article was received on 18 Jan 2017, accepted on 20 Apr 2017 and first published on 25 Apr 2017


Article type: Paper
DOI: 10.1039/C7AN00107J
Citation: Analyst, 2017, Advance Article
  • Open access: Creative Commons BY license
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    LILY-lipidome isotope labeling of yeast: in vivo synthesis of 13C labeled reference lipids for quantification by mass spectrometry

    E. Rampler, C. Coman, G. Hermann, A. Sickmann, R. Ahrends and G. Koellensperger, Analyst, 2017, Advance Article , DOI: 10.1039/C7AN00107J

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