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Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry


Bottom-up mass spectrometry–based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue disruption by ultrasonication, heat-induced antigen retrieval and two alternative methods for efficient detergent removal to enable quantitative proteomic analysis of limited amounts of FFPE material. To show the applicability of our approach, we used hepatocellular carcinoma (HCC) as a model system. By combining the described protocol with laser-capture microdissection, we were able to quantify the intra-tumor heterogeneity of a tumor specimen on the proteome level using a single slide with tissue of 10-µm thickness. We also demonstrate broader applicability to other tissues, including human gallbladder and heart. The procedure described in this protocol can be completed within 8 d.

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Fig. 1: Workflow for the proteomic analysis of microdissected FFPE specimens by quantitative mass spectrometry.
Fig. 2: Laser-capture microdissection of a hepatocellular carcinoma specimen and analysis by label-free quantitative mass spectrometry.
Fig. 3: Subsequent pooling of fractions from high-pH separation.
Fig. 4: Reproducible protein quantification from FFPE slides obtained from different tissues.
Fig. 5: Immunohistochemical validation of spatial proteomic data obtained from an FFPE tumor specimen.

Data availability

TMT and DIA data of the HCC samples shown in Figs. 4 and 5 are available via the ProteomeXchange Consortium ( or the PRIDE Proteomics Identification Database ( with the dataset identifier PXD007052. Lists of proteins identified in the experiments shown in Fig. 4 are provided in Supplementary Table 1. Raw data for gallbladder (Fig. 4e) and heart (Fig. 4f) samples are available from the authors upon request.


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The authors acknowledge I. Heinze and the CF Proteomics of the FLI for technical support; members of the tissue bank of the National Center for Tumor disease (NCT) Heidelberg, in particular E. Herpel and V. Geissler, for their support; as well as J. Scheuerer for her support with the laser microdissection; and L. Reiter and J. Muntel for critical reading of the manuscript. M.B. acknowledges funding from the European Molecular Biology Laboratory and the Max Planck Society. L.F. acknowledges financial support from project ERAatUC, grant no. 669088, under the Horizon 2020 program of the European Commission. D.S. was supported by a PhD fellowship from the Portuguese Foundation for Science and Technology (FCT, PD/BD/106051/2015) under the Inter-University Doctoral Program in Aging and Chronic Diseases. S.R. acknowledges funding from the Wilhelm Sander-Stiftung (no. 2015.111.1) and was supported in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; Project ID 314905040 – TRR 209 within Project B01). S.S. acknowledges funding from the DFG: SFB/TR209 (B04) and Si 1487/3-1, from the Hella-Buehler-Foundation and from an HRCMM (Heidelberg Research Center for Molecular Medicine) Career Development Fellowship. A.O. acknowledges funding from DFG via the Research Training Group ProMoAge (GRK 2155), the Else Kröner Fresenius Stiftung (award no. 2019_A79) and the Deutsches Zentrum für Herz-Kreislaufforschung (award no. 81X2800193). The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia.

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Authors and Affiliations



Conceptualization: K.B., J.M.K., S.S., M.B., A.O. Data analysis: K.B., J.M.K., A.O. Investigation: K.B., J.M.K., F.T., D.S. Methodology: K.B., J.M.K., S.S., M.B., A.O. Project administration: M.B., A.O. Data analysis: K.B., J.M.K., A.O. Supervision: S.R., L.F., M.B., A.O. Visualization: K.B., A.O. Writing (original draft): K.B., J.M.K., M.B., A.O. Writing (review and editing): S.S.

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Correspondence to Martin Beck or Alessandro Ori.

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Key references using this protocol

Buczak, K. et al. Mol. Cell. Proteomics 17, 810–825 (2018):

Heinze, I. et al. BMC Biol. 16, 82 (2018):

Supplementary information

Reporting Summary

Supplementary Table 1

Proteins identified in the experiments shown in Fig. 4.

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Buczak, K., Kirkpatrick, J.M., Truckenmueller, F. et al. Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry. Nat Protoc 15, 2956–2979 (2020).

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