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Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues

Abstract

Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the γ-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III–like motifs.

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Figure 1: Schematic representation of the time-controlled transcardiac perfusion cross-linking procedure.
Figure 2: Tissue integrity is an important factor in the cross-linking outcome.
Figure 3: Validation of tcTPC using γ-secretase components as protein targets.
Figure 4: Stringent purification of PrP-containing complexes after tcTPC of outbred mice.

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Acknowledgements

We thank Paul Fraser for the generous supply of aph-1–directed antibodies. N-CAM–knockout mice were generously provided by Kathryn Crossin. We also thank the Hunter's Point Animal Facility. This work was supported by grants from the National Institutes of Health (nos. AG02132 and AG010770) and a gift from the G. Harold and Leila Y. Mathers Charitable Foundation. LC/MS/MS was carried out in the UCSF Mass Spectrometry Facility, supported by National Institutes of Health grant no. NCRR RR01614.

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Correspondence to Gerold Schmitt-Ulms or Michael A Baldwin.

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S.J.D., S.B.P. and F.E.C. have financial interests in InPro Biotechnology.

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Schmitt-Ulms, G., Hansen, K., Liu, J. et al. Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues. Nat Biotechnol 22, 724–731 (2004). https://doi.org/10.1038/nbt969

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