| The structure of protein are closely related to the function of protein.Functions of proteins are performed through protein higher order structures and the protein-drugs complex formed by inter-molecular interactions.Characterization of native conformations and non-covalent complexes of proteins are of great significance to understand a variety of biological processes,drug mechanisms and drug discoveries on the molecular level.The development of mass spectrometry,especially eletrospray ionization mass spectometry(ESI-MS) technique has been widely applied in this field. However,ESI-MS technique has limitation in characterizing precisely non-covalent interaction of proteins because of its harsh ionization conditions.The objective of this paper was to solve this difficult problem.In this paper,a novel cold-spray ionization mass spectrometry(CSI-MS) method was investigated allowing the convenient and precise characterization of native conformations and non-covalent complexes of proteins. In addition,a rapid and simple CSI-MS method was developed to characterize the cluster formation of organic salt drugs.1.Characterizing native protein conformation by CSI-MS methodThe optimum conditions were determined by changing the instrumental settings including needle voltage,orificel voltage,spray temperature and experimental conditions such as solvent surface tension.A CSI-MS method was established for characterizing the native protein conformation in water-acetic acid solution.The results showed that protein CSI-MS spectra were affected by the instrumental settings.Among these parameters,the orificel voltage and spray temperature had critical effects on protein spectra.In addition, protein CSD in CSI-MS spectra did not seem to be limited by the solvent surface tension as predicted by Rayleigh equations.2.Charactering acid-induced conformational changes and non-covalent interactions by CSI-MS methodIn this paper,the equilibrium acid-induced confonnational transitions of proteins, including cytochrome c,ubiquitin,myoglobin and cyclophilin A(CypA) were investigated using CSI-MS over a wide range of pH values in aqueous solutions. Comparisons were made between the CSI-MS and traditional ESI-MS spectra. Significant narrower charged-state distribution and a shift to lower charge state were observed in CSI-MS spectra compared to ESI-MS.In addition,non-covalent complexes were observed in case of the protein-ligand complex between CypA and cyclosporin A (CsA).Furthermore,protein conformations characterized by CSI-MS were comparable with those obtained by other established biophysical methods.The results indicated CSI-MS method was a powerful tool to characterize the exactly acid-induced conformational transitions and could protect native conformations and protein complex structures from damages.3.Non-covalent interaction between amyloid-β-peptide(1-40) and Dactylorhin B by using CSI-MS methodAmyloid-β-peptide(1-40)(Aβ) is the major proteinaceous component of senile plaques formed in Alzheimer's disease(AD) brain.Aβ's aggregational properties and its complex formation ability with DHB were studied as a function of time. Furthermore,the Aβ-DHB complex's influence on Aβ's aggregation was investigated. The results showed that DHB had the ability to inhibit Aβ's aggregation which confirmed the pharmaceutical mechanism of DHB.4.Development of a CSI-MS method for the characterization of organic salt drugs.Organic salts drugs including sulfate,hydrochloride and sulfonate were selected and detected by CSI-MS.The optimal conditions were determined under different experiment conditions and instrumental settings.In the positive ion mode of CSI,the cluster ions of these salts were mainly formed at 1:1 or 2:1 ratio.This study proved that CSI-MS can be effectively characterized the overall structure of organic salt drugs.A rapid and simple CSI-MS method was developed to characterize cluster formation of organic salt drugs.
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