Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins

• DOI: 10.1021/acs.jproteome.3c00908
• Publication Date: 2024-03-20T20:59:43Z
• Authors: Karl J. Schreiber; Eileigh Kadijk; Ji-Young Youn
• Keywords: Streptavidin; Sepharose

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes streptavidin resins influence PDB results. We compared high-confidence interactors DNA/RNA-binding transactive response DNA-binding 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. also evaluated two commercial affinity for purification biotinylated proteins: conventional sepharose versus new trypsin-resistant conjugated to magnetic resin, which significantly reduces level contamination by peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled mass spectrometry in data-dependent acquisition mode, database searching, statistical analysis SAINTexpress. The APEX2-TDP-43 experiment more than miniTurbo-TDP-43, although provided greater overlap with previously documented TDP-43 interactors. Purifications on resin yielded small-scale experiments range volumes. suggest that resin-specific background binding profiles lysate-to-resin ratios cumulatively affect distributions prey abundance experimental control samples, impact confidence scores. Overall, highlight key variables consider empirical optimization experiments.