Research And Application Of Cold Spray Mass Spectrometry

The drug-protein noncovalent interaction is an important way for drugs to exert their therapeutic effect.The mode of action and the strength of binding often determine the utilization rate of drug and biological activity.Elucidating the molecular mechanism between them is crucial for understanding the drug transport process in vivo and rapid screening of candidate drugs and biomarkers.It has been an important topic in the life science,pharmacy and medicine.However,the lack of suitable analytical methods to dynamically and comprehensively evaluate the drug-protein noncovalent interaction has become a bottleneck in the development of this field.Ambient mass spectrometry(AMS)is a forefront in the domain of mass spectrometry in recent years.It has the advantages of simple installation,no sample pretreatment,on-line detection and high throughput,and greatly promotes the research process of drug-protein interaction.Especially,the "soft" ionization method of coldspray mass spectrometry(CSI-MS)can well maintain the natural conformation and weak interactions of biomolecules in solution,which makes it possible to dynamically study the noncovalent interactions between drugs and proteins.In this paper,a new method for detecting the drug-protein interactions and protein conformational changes based on CSI-MS is developed.The contents are as follows:1.A new CSI-MS method was developed to study the noncovalent interaction between phenolic acid and lysozyme(Lys).This method can not only truly characterize the noncovalent complex,but also obtain the information of protein conformational changes and detailed binding parameters.It provides data support for the comprehensive evaluation of the binding process of phenolic acid and Lys and the indepth elucidation of the biological activities of phenolic acid.The stragetry that combined with CSI-MS and MS/MS techniques,multispectrometry and theoretical calculation were used to establish the characterization method of structure composition,stability and the binding affinity between phenolic acid and Lys in the gas phase.The order of the binding affinity between phenolic acid and Lys(4,5-CQA > RA > 1,3-CQA > CQA)and the binding constants were obtained.Mowerve,the bingding affinity in the liquid phase was determined by the static quenching mechanism of fluorescence spectrum,which was consistent with the gas phase detection.The CD and FTIR spectra revealed that phenolic acid changed the secondary structure of Lys and increased the ?-helix content,indicating an increase in the tryptophan(W)hydrophobicity near the protein binding site resulting in a conformational alteration of the protein.Molecular docking studies further revealed the binding sites and binding modes,which supported the experimental results of CSI-MS.All the above results show that the CSI-MS method is reliable and can provide a new technical support for the study of mechanism of drugs.2.A new CSI-MS method for studying the oligomers of islet amyloid polypeptide(IAPP)and the noncovalent interaction between IAPP and its inhibitors was established.This method can not only realize the online detection and structure analysis of IAPP oligomers,but also can be used for rapid screening of IAPP-inhibitors in vitro.It provides a new technique for drug development of type 2 diabetes.The CSI-MS technology was employed to characterize the oligomers for the first time.The comparison with ESI-MS proved that the conditions of CSI were softer and the effects of different experimental conditions(ionic strengths,organic solvent and p H)on the determination of oligomers and protein conformational stability were investigated.The CID-MS/MS experiments showed that odd charge dimer ions of IAPP were composed of two monomer products and was mainly noncovalent dissociation.In addition,we first explored to establish a CSI-MS method for screening IAPPinhibitors and applied it to the study of flavonoids-inhibitors,from which we screened out the two most promising inhibitors(rutin and quercitrin).Then,the noncovalent binding mechanism and structure-activity relationship between flavonoids and IAPP were deeply studied.The results showed that 3-OH and sugar chain in the structure of flavonoids were the active groups,and hydrogen bonds played a major role in the binding.Furthermore,fluorescence spectroscopy and TEM experiments were used to further verify that the screened rutin and quercitrin could effectively inhibit the fibers formation of IAPP,which proved the accuracy and reliability of CSI-MS method.This study expanded the application of CSI-MS in the development of amyloid polymers and its inhibitors,and provided a new insight to elucidate the anti-amyloid mechanism of flavonoids.