TY - JOUR
T1 - LILY-lipidome isotope labeling of yeast: in vivo synthesis of 13C labeled reference lipids for quantification by mass spectrometry
AU - Rampler, Evelyn
AU - Coman, Cristina
AU - Hermann, Gerrit
AU - Sickmann, Albert
AU - Ahrends, Robert
AU - Koellensperger, Gunda
N1 - Publisher Copyright:
© The Royal Society of Chemistry 2017.
PY - 2017/6/7
Y1 - 2017/6/7
N2 - 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 10
8 yeast cell starting material; RSDs <10% (N = 4)).
AB - 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 10
8 yeast cell starting material; RSDs <10% (N = 4)).
KW - POLYUNSATURATED FATTY-ACIDS
KW - PICHIA-PASTORIS
KW - SHOTGUN LIPIDOMICS
KW - HUMAN PLASMA
KW - QUANTITATIVE-ANALYSIS
KW - INTERNAL STANDARD
KW - AMINO-ACIDS
KW - PROTEIN
KW - METABOLOMICS
KW - REVEALS
UR - http://www.scopus.com/inward/record.url?scp=85021733440&partnerID=8YFLogxK
U2 - 10.1039/c7an00107j
DO - 10.1039/c7an00107j
M3 - Article
SN - 0003-2654
VL - 142
SP - 1891
EP - 1899
JO - The Analyst
JF - The Analyst
IS - 11
ER -