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Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Nature Protocols volume 6, pages 1695–1709 (2011)Cite this article

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Abstract

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.

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Figure 1: Workflow for investigation of peptide content in FFPE-TMA.
Figure 2: Trypsin and matrix application.
Figure 3: Protein identification workflow.
Figure 4: Portrait spotting setup.
Figure 5: IMS correlation with pathological diagnosis.
Figure 6: MALDI images of three tryptic peptides originating from heat shock protein β-1 detected in two of three squamous cell carcinoma core biopsies analyzed in a TMA.

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Acknowledgements

We thank P.M. Angel, M.L. Reyzer and E.H. Seeley for helpful discussions and critical reviewing of the manuscript. We also thank J.L. Allen, E.H. Seeley and K. Schwamborn for development and optimization of the deparaffinization and antigen retrieval methods, and M. Reid Groseclose for providing some TMA images and the figures in the Anticipated Results section. This work was supported by the US National Institutes of Health/National Institute of General Medical Sciences grants 5RO1GM058008-11 and DOD W81XWH-05-1-0179.

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  1. Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, USA

    Rita Casadonte & Richard M Caprioli

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Both authors contributed equally to the development of the protocol and the writing of the manuscript.

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Correspondence to Richard M Caprioli.

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Casadonte, R., Caprioli, R. Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry. Nat Protoc 6, 1695–1709 (2011). https://doi.org/10.1038/nprot.2011.388

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