Study of the noncovalent interactions between phenolic acid and lysozyme by cold spray ionization mass spectrometry (CSI-MS), multi-spectroscopic and molecular docking approaches.

Elucidating the recognition mechanisms of the noncovalent interactions between pharmaceutical molecules and proteins is important for understanding drug delivery in vivo, and for the further rapid screening of clinical drug candidates and biomarkers. In this work, a strategy based on cold spray ionization mass spectrometry (CSI-MS), combined with fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and molecular docking methods, was developed and applied to the study of the noncovalent interactions between phenolic acid and lysozyme (Lys). Based on the real characterization of noncovalent complex, the detailed binding parameters, as well as the protein conformational changes and specific binding sites could be obtained. CSI-MS and tandem mass spectrometry (MS/MS) technique were used to investigate the phenolic acid-Lys complexes and the structure-affinity relationship, and to assess their structural composition and gas phase stability. The binding affinity was obtained by direct and indirect MS methods. The fluorescence spectra showed that the intrinsic fluorescence quenching of Lys in solution was a static quenching mechanism caused by complex formation, which supported the MS results. The CD and FTIR spectra revealed that phenolic acid changed the secondary structure of Lys and increased the α-helix content, indicating an increase in the tryptophan (W) hydrophobicity near the protein binding site resulting in a conformational alteration of the protein. In addition, molecular docking studies were performed to investigate the binding sites and binding modes of phenolic acid on Lys. This strategy can more comprehensively and truly characterize the noncovalent interactions and can guide further research on the interactions of phenolic acid with other proteins.
Competing Interests: Declaration of competing interest The authors declare no competing financial interest.
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