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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...

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Hauptverfasser: Buczak, Katarzyna (Verfasst von) , Kirkpatrick, Joanna M. (Verfasst von) , Truckenmüller, Felicia (Verfasst von) , Santinha, Deolinda (Verfasst von) , Ferreira, Lino (Verfasst von) , Rössler, Stephanie (Verfasst von) , Singer, Stephan (Verfasst von) , Beck, Martin (Verfasst von) , Ori, Alessandro (Verfasst von)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 31 July 2020
In: Nature protocols
Year: 2020, Jahrgang: 15, Heft: 9, Pages: 2956-2979
ISSN:1750-2799
DOI:10.1038/s41596-020-0356-y
Online-Zugang:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1038/s41596-020-0356-y
Verlag, lizenzpflichtig, Volltext: https://www.nature.com/articles/s41596-020-0356-y
Volltext
Verfasserangaben:Katarzyna Buczak, Joanna M. Kirkpatrick, Felicia Truckenmueller, Deolinda Santinha, Lino Ferreira, Stephanie Roessler, Stephan Singer, Martin Beck and Alessandro Ori
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Zusammenfassung: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.
Beschreibung:Gesehen am 11.06.2026
Beschreibung:Online Resource
ISSN:1750-2799
DOI:10.1038/s41596-020-0356-y

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