Automated AssayMap Workflow for Proximity-Dependent Labeling Coupled to Mass Spectrometry Proteomics
Proximity-dependent labeling followed by Affinity Purification coupled with Mass Spectrometry (AP–MS) analysis for identifying protein-protein interactions (PPIs) have become an established approach utilized by hundreds of researchers within the last decade. In general proximity-dependent labeling methods involve the expression of a bait protein fused with the promiscuous biotin ligase. BioID or TurboID technologies use biotin and ATP as substrates to generate a highly reactive biotinyl-5′-AMP intermediate that, in turn, labels primary amines on nearby (labeling radius to ~10 nm) proteins (on lysine side chains). A typical sample preparation workflow following in-cell biotylation involves cell lysis, pulldown of biotinylated proteins using streptavidin beads, washes to remove background, digestion of bound proteins directly on beads and finally desalt of digested peptides. Most labs carry out this workflow manually which will require experienced laboratory personnel, and even for a seasoned staff, there are other potential downsides of a manual workflow such as low throughput, poor reproducibility and a higher risk of errors.
In this presentation I will show a fully automated streptavidin AP-MS preparation for any proximity-dependent labeling (i.e., BioID, TurboID, APEX, etc) protocol using the AssayMap Bravo platform. Our protocol starts with cell lysate and output peptides for mass spec, and it is highly-sensitive and reproducible. In addition, we show a high-throughput in preparation samples for mass spec, with 96 samples prepared in less than 7 hours. I will also show the use of our automated AssayMap workflow in a recent BioID study aimed to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins.
Presenter: Alex Rosa Campos (Director – Proteomics, Sanford Burnham Presbys Medical Discovery Institute)
Alex earned his Ph.D. from the University of Barcelona (Spain), where he worked in the mass spectrometry laboratory at the Barcelona Science Park and gained extensive experience in mass spectrometry, proteomics and bioinformatics. He then served as project manager at Integromics leading a group of computational scientists and software developers. Alex was recruited to SBP in 2013 as postdoc in a joint initiative between MedImmune and the SBP, and became director of the Proteomics Core in 2016 with the launch of the SBP Proteomics Center of Excellence. Alex manages scientific projects and researchers, and oversees the day-to-day operation of the personnel. He is also in charge of all proteomics data mining and has an active role in proteomics method development in the laboratory and in collaboration with other research groups in the academia and industry.
