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

TL;DR

PUB-MS is a novel mass spectrometry-based technique that detects protein-protein proximity in vivo by biotinylation, enabling multiplex and temporal analysis in standard proteomics labs.

Contribution

It introduces a redesigned BAP sequence for easy detection and demonstrates the method's specificity, multiplexing, and temporal capabilities in various experimental models.

Findings

Biotinylation is specifically enhanced when proteins are in proximity.

Mass spectrometry allows multiplex analysis with different BAP sequences.

Method can study specific protein subfractions at defined times.

Abstract

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.