Talk title: Improved sensitivity and specificity of proximity-dependent biotinylation approaches for interactomics
Akhilesh Pandey, M.D., Ph.D.
Johns Hopkins University School of Medicine, Baltimore
Dr. Akhilesh Pandey is currently a Professor at the McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Oncology and Pathology at the Johns Hopkins University School of Medicine. He is also the Founder and Director of the Institute of Bioinformatics, a non-profit research institute in Bangalore, India and a Wellcome Trust/ Department of Biotechnology India Alliance Fellow. He has pioneered methods for quantitative proteomics (SILAC), for analysis of post-translational modifications and for proteogenomics analyses. Dr. Pandey’s laboratory is taking a systems biology approach by combining many ‘Omics’ technologies to study signal transduction pathways in health and disease.
Proteins rarely function in isolation as they often occur as components of multiprotein complexes. Mass spectrometry-based proteomic approaches offer an opportunity to identify protein-protein interactions in an in vivo setting in a sensitive fashion. However, a major limitation of immunoprecipitation-based strategies for identifying protein interactors is that it is difficult to detect transient interactions as well as interactors that are present in low abundance in cells. To circumvent this, proximity-dependent biotinylation strategies (e.g. BioID) are being increasingly used where the bait protein is fused to a promiscouous biotin ligase, BirA, leading to biotinylation of the interacting proteins. In this approach, the biotinylated proteins are generally identified by digesting proteins captured by streptavidin beads which leads to identification of proteins although biotinylated peptides are not directly identified. We have recently developed a method for direct capture of biotinylated peptides which increases both the sensitivity and specificity of experiments that employ this biotinylation-based approach. I will discuss our recent studies using this strategy for interactome analysis using isogenic systems for analysis of signaling pathways.