  The common techniques to study protein-protein proximity in vivo are not
well-adapted to the capabilities and the expertise of a standard proteomics
laboratory, typically based on the use of mass spectrometry. With the aim of
closing this gap, we have developed PUB-MS (for Proximity Utilizing
Biotinylation and Mass Spectrometry), an approach to monitor protein-protein
proximity, based on biotinylation of a protein fused to a biotin-acceptor
peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The
biotinylation status of the BAP can be further detected by either Western
analysis or mass spectrometry. The BAP sequence was redesigned for easy
monitoring of the biotinylation status by LC-MS/MS. In several experimental
models, we demonstrate that the biotinylation in vivo is specifically enhanced
when the BAP- and BirA- fused proteins are in proximity to each other. The
advantage of mass spectrometry is demonstrated by using BAPs with different
sequences in a single experiment (allowing multiplex analysis) and by the use
of stable isotopes. Finally, we show that our methodology can be also used to
study a specific subfraction of a protein of interest that was in proximity
with another protein at a predefined time before the analysis.
