Abstract
The time-controlled transcardiac perfusion crosslinking (tcTPC) method differs from conventional perfusion fixation in that the crosslinking reagent is administered throughout the circulatory system for only a relatively short period of time, thereby allowing limited crosslinking to occur. Bait protein complexes are isolated by affinity capture (AC) under stringent conditions and are recovered from the AC matrix by acidic elution. Affinity-purified proteins are reduced, alkylated, and digested with a specific endoproteinase, such as trypsin. Subsequently, peptides are isotopically labeled, separated by reversed-phase chromatography and analyzed by quantitative tandem mass spectrometry (MS/MS). The proteins crosslinked to the bait protein during tcTPC are identified by database searches with conventional protein identification software. The tcTPC strategy offers unique advantages over alternative approaches for studying a subset of protein complexes which require a particular environment for their structural integrity, such as membrane protein complexes that are notorious for their tendency to dissociate upon detergent solubilization. The sensitivity and utility of this method are influenced by the spatial distribution of chemical groups within the bait protein complexes that can engage in productive crosslinks.
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Acknowledgments
Work on this project was funded through an operating grant MOP-74734 of the Canadian Institute for Health Research (CIHR). AJ was supported by an award from The Scottish Rite Charitable Foundation of Canada and GS received support from the W. Garfield Weston Foundation.
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Jeon, A.H.W., Schmitt-Ulms, G. (2012). Time-Controlled Transcardiac Perfusion Crosslinking for In Vivo Interactome Studies. In: Drewes, G., Bantscheff, M. (eds) Chemical Proteomics. Methods in Molecular Biology, vol 803. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-364-6_16
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