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The common techniques to study protein-protein proximity in vivo are notwell-adapted to the capabilities and the expertise of a standard proteomicslaboratory, typically based on the use of mass spectrometry. With the aim ofclosing this gap, we have developed PUB-MS (for Proximity UtilizingBiotinylation and Mass Spectrometry), an approach to monitor protein-proteinproximity, based on biotinylation of a protein fused to a biotin-acceptorpeptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. Thebiotinylation status of the BAP can be further detected by either Westernanalysis or mass spectrometry. The BAP sequence was redesigned for easymonitoring of the biotinylation status by LC-MS/MS. In several experimentalmodels, we demonstrate that the biotinylation in vivo is specifically enhancedwhen the BAP- and BirA- fused proteins are in proximity to each other. Theadvantage of mass spectrometry is demonstrated by using BAPs with differentsequences in a single experiment (allowing multiplex analysis) and by the useof stable isotopes. Finally, we show that our methodology can be also used tostudy a specific subfraction of a protein of interest that was in proximitywith another protein at a predefined time before the analysis.

PUB-MS - a mass-spectrometry-based method to monitor protein-protein proximity in vivo