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Neef, S.K.* ; Winter, S.* ; Hofmann, U.* ; Mürdter, T.E.* ; Schaeffeler, E.* ; Horn, H.* ; Buck, A. ; Walch, A.K. ; Hennenlotter, J.* ; Ott, G.* ; Fend, F.* ; Bedke, J.* ; Schwab, M.* ; Haag, M.*

Optimized protocol for metabolomic and lipidomic profiling in formalin-fixed paraffin-embedded kidney tissue by LC-MS.

Anal. Chim. Acta 1134, 125-135 (2020)

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Open Access Green as soon as Postprint is submitted to ZB.

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Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines LPED remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Formalin-fixed Paraffin-embedded Kidney Tissue ; Metabolite Extraction ; Metabolomics ; Lipidomics ; Non-targeted Metabolomics ; Mass Spectrometry; Performance Liquid-chromatography; Mass-spectrometry; Cancer; Brain; Formaldehyde; Extraction; Propofol; Dna; Recommendations; Metabolites

ISSN (print) / ISBN 0003-2670

e-ISSN 1873-4324

Journal Analytica Chimica Acta

Quellenangaben Volume: 1134, Pages: 125-135

Publisher Elsevier

Publishing Place Radarweg 29, 1043 Nx Amsterdam, Netherlands

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Research Unit Analytical Pathology (AAP)

Grants Deutsche Krebshilfe
Deutsche Forschungsgemeinschaft
Ministry of Education and Research of the Federal Republic of Germany (BMBF)
ICEPHA Graduate Program, University of Tubingen (Tubingen, Germany)
Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy
Robert Bosch Stiftung (Stuttgart, Germany)