Protein affinity selection method utilizing mass spectrometry has now become an invaluable tool for identification of protein binding partners. Although the affinity-directed mass spectrometry analytical platform for selectively capturing interacting proteins has been implemented to discover many missing ligands, determination of true binding partners from co-purified proteins in the affinity-purified sample is extremely challenging. The challenge can be addressed by biophysical characterizations of newly identified binding partners prior to characterizing their biological associations. Several biophysical characterization methods such as (i) immunoassays (ii) molecular binding simulation (iii) mass spectrometry-based binding epitope mapping and (iv) peptide microarray-based binding epitope mapping can be implemented to evaluate the binding interaction. Herein, adapting the affinity-directed mass spectrometry method, we identified several potential ligands of a specific protein expressed on the surface of immune cells. Among them, we selected one immune cell-specific binding protein as a ligand based on its abundance. We further evaluated the binding interaction between the immune cell expressed protein and its selected ligand using the biophysical assays and confirmed that the selected ligand is specifically interacting with an immune cell specific expressed protein. The affinity-directed mass spectrometry analytical platform implemented in this investigation is expected to be of general use and to facilitate more accurate identification of many missing ligands involved in various biological processes.
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Analyzing biomolecular interactions by affinity-directed mass spectrometry
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