2011/08/29 by Arman Kulyyassov, Muhammad Shoaib, Kulyyassov, Arman +11
Biochemistry, Genetics and Molecular Biology · Medicine · #Biotin and Related Studies #Cellular transport and secretion #FOS: Biological sciences #Molecular Networks (q-bio.MN) #Peptidase Inhibition and Analysis
paper · pdf · doi:10.48550/arxiv.1108.5657
openalex publication_date 2011/08/29 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28
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.