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In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature

Abstract

Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes.

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Figure 1
Figure 2: Experimental setup of in vivo perfusion.

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Acknowledgements

We are grateful to the Functional Genomics Center Zurich for access to mass spectrometers and support. Financial support from the EU project STROMA, the Bundesamt für Bildung und Wissenschaft (FLUOR-MMPI and STROMA), the Swiss National Science Foundation and the Gebert-Rüf Foundation are gratefully acknowledged.

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The study was designed and the paper written by D.N., C.R. and J.-N.R.; experiments were performed by J.-N.R. and C.R.

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Correspondence to Christoph Roesli or Jascha-N Rybak.

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The authors declare no competing financial interests.

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Roesli, C., Neri, D. & Rybak, JN. In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature. Nat Protoc 1, 192–199 (2006). https://doi.org/10.1038/nprot.2006.29

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