| The main contributions of this dissertation for the degree of doctor of philosophy are listed as followed:Using ESI-MS, studying a typical nonapeptide and the non-covalent interaction with about20kinds of metal ions systemly. Analysing the differences between peptide-metal ions non-covalent complexes. Using a program self-compiled to calculate the m/z of fragment ions and an innovative analytical system to rank the fragment ions of doubly-charged complexes with more than one metal ion especially. Explaining the metal-induced conformation change in the combination process Pointing out the selectivity of combination sites with amino acid residues by different metal ions, and the main factor to influence the site-selectivity. Subsequently, the study extended to a typical protein molecule. Studying the the solution environment-induced conformation change by changing different solution pH or alcohol rate, and the protein fragmentation of energy, protein-metal ions non-covalent binding.Mass Spectrometry Identification of the material composition analysis and has high sensitivity, high resolution, is currently one of the most widely used analytical technologies. ESI-MS can be more moderate to produce multiple charged gas ions, not only get accurate molecular weight, but also can be very suitable for studying weak bond interactions. With application of collision induced dissociation, ESI-MS can also be used for structural analysis, reaction mechanism, non-covalent interaction and so on. Peptides and proteins are the most important part of organisms. Their structural changes, often accompanied by changes in biological function, leading to the phenomenon of life, such as health status changes. In recent years, with the rapid development of proteomics, peptides and proteins have also been a more widespread concern. relevant research has been used for disease prediction and drug synthesis, made major achievements. ESI-MS of its own characteristics, has become the undisputed the most important tools for the studying of peptide and protein molecules.Paradox protein folding problem have been proposed since1969and have not yet cracked today. So changes on the protein conformation remains an important research topic. In addition, the peptide and protein can bind with metal ions to form non-covalent complexes which is also an important part of proteomics, playing an important role in the life course. Therefore, this article select to study the metal ions-induced conformational change of peptide as the main subject of this topic. Using collision induced dissociation, a detailed study of the relevant binding and conformational change of the process have also been made. Peptide is a low-level structure of the protein molecules, the results can provide important and reliable basic information for such studies of protein. This article also discusses a variety of different solution environment-induced conformation changes of protein, including the secondary structure and tertiary structure. Finally, we do some preliminary research on the protein dissociation and non-covalent interactions between protein and metal ions.This doctoral dissertation consists of six parts and the contents are summarized as follows:Chapter One:Introduction. This chapter outlines the basic principles of ESI-MS and applications that use the technique to study molecular structure and composition which is very import. The paper highlights the composition and structure of peptide and protein molecules, also described the pH, solvent composition, metal ions can induce conformational changes of such biological molecules. Application of ESI-MS can analyze the conformation changes of biological molecules binding with a variety of ligands to form non-covalent complexes. Based on these comments and summary of Chapter One, research presented in this paper and research ideas were made.Chapter Two:Studying on bradykinin ions and its fragments. Bradykinin is a nonapeptide with important physiological functions, containing almost all types of amino acids and having a relatively simple spatial structure. It is suitable to do subtle conformational study. Four different kinds of Mass spectrometers were used to do the research on bradykinin. Based on their performance, ESI Q-Tof MS was selected to do a detailed study on bradykinin ions and its fragments. First, we can get the three kinds bradykinin ions, and then we choose singly-charged ion and doubly-charged ion as parent ions to get their daughter ions information. According to the theoretical calculation and analysis of the possible peptide fragments, we get the pieces in accordance with sub-ion mass spectrometry peaks, respectively, were assigned to their analysis. Changing the collision energy from OeV to100eV, obtaining the fragmentation process of the two ions. Doubly-chaged ion can provide more sufficient structural information. Singly-charged ion need more collision energy to dissociation, and get mainly y-type ions.Chapter Three:Studying on bradykinin-alkali metal non-covalent complexes. Alkali metal ions has a variety important biological function. The binding site and alkali metal ions-induced conformation change of peptide is still controversial. We fixed with the same experimental conditions as last chapter, using single-stage MS and tandem mass spectrometry to study the non-covalent interaction between bradykinin to alkali metal ions. The results showed that the two main complex ions were formed as [BK+(2-n) H+nM]2+and [BK+(1-n) H+nM]+(0≤n≤4). Alkali metals shows different combination capabilities, according to the strength of the order are:Sodium ion, Lithium ion, potassium ion, cesium and rubidium ions. With an innovative analytical method to compared with different number of metal ion attached to bradykinin, complexes were analyzed and found that alkali metal ions had different selectivity to choose amino acid residues. The dissociation process can be divided into three categories. Electronegativity of alkali metal, conformation of bradykinin ions, alkali metal ion radius, and the mass number all determine their amono acid residue selectivity. Alkali metal ions will induce the conformation change of bradykinin.Chapter Four:Studying on bradykinin-transition metal non-covalent complexe. Transition metal ions has unsaturated d-electron, often with special properties and toxic. Since non-covalent binding ability of transition metal ions are often weak, so studies were less. Choose thirteen transition metal ions containing the same period, with the family and a variety of valence states to use single-stage MS and tandem mass spectrometry to study the non-covalent interaction between transition metal ions and bradykinin. The results show that monovalent ions has similar binding situation with alkali metal ions, divalent ion is slightly different to form [BK+(1-2n) H+nM]+and [BK+(2-2n) H+nM]2+(0≤n≤4), the combination of trivalent ions are very special to form doubly-charged non-covalent complexes with two bradykinin ions, however, the doubly-charged complexes are unstable. The first transition period metal ions can be divided into two categories, Cu2+and the remaining four. Transition metal ions can be combined with the peptide fragments. PtCl4clusters can be combined until added six Cl, MoCl3and RhCl3combination is none. The coordination bonds may suppress the bradykinin ions’ signal. Selectivity of transition metal ions was determined mainly by the d electron.Chapter Five:Studying on the conformation change of Myoglobin. Myoglobin is an important oxygen storage material. We use ESI-MS, CD and UV-Vis spectrum to study the solution environment-induced conformation change by changing the pH value, respectively, and the solvent composition. When the pH=4.0, we will30%alcohol volume rate, use six different alcohol to induce Myoglobin conformation change. The results show the conformation of myoglobin molecule changed, both the iron coordination and spin state. When the solvent is methanol, we change the solution pH as11.0,11.4,12.0, with different volume fractions (0%,10%,30%,50%,70%,90%) of methanol. The results show that, pH induced secondary structure of protein, the content of methanol induced the tertiary structure changes. pH=11.4,30%methanol is a degenerative knee condition. Preliminary study of the fragmentation energy of Myoglobin and non-covalent binding with metal ions were also made. We found that hMb mainly lose h while aMb had a more complicated fragment mode, and aMb15+is unique with the lowest energy. Myoglobin can bind with a variety of transition metal ions.Chapter Ⅵ:Summary and Outlook. The achievements of this dissertation were concluded. Meanwhile, some drawbacks in our current researches were pointed out and the corresponding improvement were also presented. |
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