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Analyst. 2017 Jun 7;142(11):1891-1899. doi: 10.1039/c7an00107j. Epub 2017 May 5.

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

Author information

1
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währingerstr. 38, 1090 Vienna, Austria. gunda.koellensperger@univie.ac.at and Vienna Metabolomics Center (VIME), University of Vienna, Althanstraße 14, 1090 Vienna, Austria and Chemistry Meets Microbiolgy, Althanstraße 14, 1090 Vienna, Austria.
2
Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany.
3
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währingerstr. 38, 1090 Vienna, Austria. gunda.koellensperger@univie.ac.at and ISOtopic Solutions, Währingerstr. 38, 1090 Vienna, Austria.
4
Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany and College of Physical Sciences, University of Aberdeen, Department of Chemistry, AB24 3UE Aberdeen, UK and Medizinische Fakultät, Medizinische Proteom-Center (MCP), Ruhr-Universität Bochum, 44801 Bochum, Germany.

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)).

PMID:
28475182
DOI:
10.1039/c7an00107j
[Indexed for MEDLINE]

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