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In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature

Nature Methods volume 2, pages 291–298 (2005)Cite this article

A Corrigendum to this article was published on 01 May 2005

Abstract

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography–tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.

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Figure 1: Schematic representation of the relevant steps of the in vivo biotinylation method.
Figure 2: Histochemical and immunofluorescence analysis of organ and tumor sections.
Figure 3: Chromatographic elution profiles of tryptic peptides from the healthy and tumor-bearing kidneys of three independent RENCA mice.
Figure 4: Western blot analysis of organ-specific markers (a) Equal amounts of heart, kidney, liver, skeletal muscle, F9 and RENCA tumor total protein extracts from a saline-perfused SvEv mouse were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.
Figure 5: Immunofluorescence detection of organ-specific markers.

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Acknowledgements

The authors are grateful to M. Le Hir (Anatomy Institute, University of Zurich) for help in the vascular perfusion fixation of mice used in indirect immunofluorescence experiments and to A. Garofalo (Mario Negri Institute) for technical support. Financial support of the Gebert-Rüf Foundation, the Swiss National Science Foundation and the Bundesamt für Bildung und Wissenschaft (European Union Projects Angiogenesis and Stroma FPS/6 #503233) is gratefully acknowledged. J.-N.R. is a recipient of a Bundesamt für Bildung und Wissenschaft salary (FLUOR–MMPI). G.E. is on leave of absence from the Institute of Neurobiology and Molecular Medicine CNR, Rome, Italy.

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Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Strasse 10, Zurich, CH-8093, Switzerland

    Jascha-N Rybak, Anna Ettorre, Dario Neri & Giuliano Elia

  2. Anatomy Institute, University of Zurich, Winterthurerstrasse 190, Zurich, CH-8057, Switzerland

    Brigitte Kaissling

  3. Department of Oncology, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, Bergamo, I-24125, Italy

    Raffaella Giavazzi

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Correspondence to Dario Neri.

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Supplementary information

Supplementary Fig. 1

Subcellular localization of proteins identified (PDF 363 kb)

Supplementary Table 1

Complete list of proteins identified in mouse organs and tumors (PDF 165 kb)

Supplementary Methods (PDF 104 kb)

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Rybak, JN., Ettorre, A., Kaissling, B. et al. In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature. Nat Methods 2, 291–298 (2005). https://doi.org/10.1038/nmeth745

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