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

Nature Protocols volume 11, pages 1428–1443 (2016)Cite this article

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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.

Author information

Author notes

  1. Alice Ly and Achim Buck: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany

    Alice Ly, Achim Buck, Na Sun, Karin Gorzolka, Annette Feuchtinger, Michaela Aichler & Axel Walch

  2. Maastricht MultiModal Molecular Imaging Institute (M4I), Maastricht University, Maastricht, the Netherlands

    Benjamin Balluff

  3. Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany

    Klaus-Peter Janssen

  4. Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands

    Peter J K Kuppen & Cornelis J H van de Velde

  5. Institute of Pathology, Technische Universität München, Munich, Germany

    Gregor Weirich, Franziska Erlmeier & Rupert Langer

  6. Institute of Pathology, Helmholtz Zentrum München, Neuherberg, Germany

    Michaela Aubele

  7. Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, Neuherberg, Germany

    Horst Zitzelsberger

  8. Centre for Proteomics and Metabolomics, Leiden University Medical Centre, Leiden, the Netherlands

    Liam McDonnell

  9. Fondazione Pisana per la Scienza ONLUS, Pisa, Italy

    Liam McDonnell

Authors
  1. Alice Ly
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  2. Achim Buck
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  3. Benjamin Balluff
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  4. Na Sun
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  5. Karin Gorzolka
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  6. Annette Feuchtinger
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  7. Klaus-Peter Janssen
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  8. Peter J K Kuppen
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  9. Cornelis J H van de Velde
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  10. Gregor Weirich
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  11. Franziska Erlmeier
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  12. Rupert Langer
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  13. Michaela Aubele
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  14. Horst Zitzelsberger
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  15. Liam McDonnell
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  16. Michaela Aichler
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  17. Axel Walch
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Contributions

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.

Corresponding author

Correspondence to Axel Walch.

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The authors declare no competing financial interests.

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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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