Confirmation of the Binding Site of Cyclophilin-Cyclosporin Non‐Covalent Complex Using Limited Proteolysis and Liquid Chromatography-Quadrupole‐Time‐of‐Flight Mass Spectrometry

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The limited proteolysis approach has been based on the fact that exposed regions of a protein can be subjected to time resolved cleavage with specific proteases. Although this is not a new concept, the ability to utilize modern mass spectrometry techniques in conjunction with limited proteolysis has gained prominence. Herein, we report the use of time‐resolved limited proteolysis, liquid chromatography, and a hybrid quadrupole‐time‐of‐flight mass spectrometry (LC‐Q‐ToF‐MS), which offers sensitivity, high mass resolution, and mass accuracy, to confirm the binding pocket of human cyclophilin A—cyclosporin A (CyP:CsA) non‐covalent complex. The hydrophobic binding pocket of human CyP is defined by 13 amino acid residues that are in contact of less than 4 Å with a segment of CsA. The effect of CsA binding on proteolytic susceptibility of CyP was evident. In the absence of CsA, a marker tryptic peptide resulting from the putative cleavage of lysines 91 and 119 was identified. This peptide contained five of the critical residues in the CyP binding pocket. Full protection against trypsin mediated cleavage at lysines 91 and 119 was achieved in the presence of CsA. The utility of this approach could be further realized in higher‐throughput structural screening of libraries of ligands for CyP.