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High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue

Abstract

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected 1,500 m/z species in the mass range m/z 50–1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

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Figure 1: Important types of FFPE tissue specimens that can be used for metabolite imaging with MALDI-MSI.
Figure 2: Workflow for the analysis of FFPE metabolite content using MALDI-MSI.
Figure 3: Comparison of m/z localization in colon cancer from a fresh frozen tissue sample and a multipatient FFPE TMA.
Figure 4: Reproducibility of the MALDI-MSI approach for FFPE metabolite analysis.
Figure 5: MSI of metabolites in a liver biopsy featuring cirrhosis.
Figure 6: Analysis of metabolite MSI data identifies survival status and a new independent cancer prognostic marker.

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Acknowledgements

This project was funded by the Ministry of Education and Research of the Federal Republic of Germany (BMBF; grant number 01ZX1310B), ERA-NET TRANSCAN-2-ARREST and the Deutsche Forschungsgemeinschaft (grant nos. HO 1254/3-1, SFB 824 TP Z02 and WA 1656/3-1). The authors thank U. Buchholz, C.-M. Pflüger, G. Mettenleiter and A. Voss for technical assistance, and D. Borgmann for bioinformatics assistance.

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A.L. and A.B. were responsible for the MALDI imaging mass spectrometry data acquisition and data analyses, and wrote the manuscript. B.B. was involved in data and bioinformatics development, and wrote the manuscript. N.S. and K.G. conducted protocol validation experiments. A.F. and K.-P.J. assisted in interpretation of the results. K.-P.J., P.J.K.K., C.J.H.v.d.V., G.W., F.E., R.L. and M. Aubele provided samples and assisted in histological interpretation of the samples. L.M. was involved in data and bioinformatics development. H.Z. and M. Aichler assisted in interpretation of the results and writing of the manuscript. A.W. conceived the study, and assisted in interpretation of results and writing of the manuscript.

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Correspondence to Axel Walch.

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Ly, A., Buck, A., Balluff, B. et al. High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue. Nat Protoc 11, 1428–1443 (2016). https://doi.org/10.1038/nprot.2016.081

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