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

被引:47
作者
Rampler, Evelyn [1 ,2 ,3 ]
Coman, Cristina [4 ]
Hermann, Gerrit [1 ,5 ]
Sickmann, Albert [4 ,6 ,7 ]
Ahrends, Robert [4 ]
Koellensperger, Gunda [1 ,2 ,3 ]
机构
[1] Univ Vienna, Fac Chem, Inst Analyt Chem, Wahringerstr 38, A-1090 Vienna, Austria
[2] Univ Vienna, Vienna Metabol Ctr VIME, Althanstr 14, A-1090 Vienna, Austria
[3] Chem Meets Microbiol, Althanstr 14, A-1090 Vienna, Austria
[4] Leibniz Inst Analyt Wissensch ISAS eV, Otto Hahn Str 6b, D-44227 Dortmund, Germany
[5] ISOtop Solut, Wahringerstr 38, A-1090 Vienna, Austria
[6] Univ Aberdeen, Dept Chem, Coll Phys Sci, Aberdeen AB24 3UE, Scotland
[7] Ruhr Univ Bochum, MCP, Med Fak, D-44801 Bochum, Germany
关键词
PICHIA-PASTORIS; QUANTITATIVE-ANALYSIS; SHOTGUN LIPIDOMICS; HUMAN PLASMA; AMINO-ACIDS; REVEALS; PROTEIN; METABOLITES; EXTRACTION; MS/MS;
D O I
10.1039/c7an00107j
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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 C-13 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 C-13 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 C-13 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)).
引用
收藏
页码:1891 / 1899
页数:9
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