Investigation Of Peptide Charge-remote Fragmentation In ESI-MS

Gas-phase ion chemistry has played a central role in mass spectrometry since its inception. However, in comparasion with rapid innovation of technologies and extensive applications of mass spectrometry fragmentation mechanism studies are far behind. This makes a restriction to take full use of MS/MS spectra for obtaining comprehensive structure information, and may also cause problems in accurate interpretation of mass spectra. At present, tandem mass spectrometry has become indipensible means in proteomic researches. In order to unambiguous identifications of peptide/protein, it is important to study and understand peptide fragmentation mechanism. In this thesis, we explore formation mechanisms of a series of unusual fragment ions generated from a model peptide Dh HP-6. To investigate possible effect of particular amino acid on the fragmentation, a series of peptide derivatives are synthesized and studied by ESI-Q-Tof. The charge-remote peptide fragmentation pathways are proposed and supported by DFT quantum calculations. The thesis is composed of five chapters; the main contents of each chapter are as follow:In chapter 1, we give an introduction of mass spectrometry based stratgies for protein identification and summarize main progresses of peptide fragmentation in recent 30 years. According to the literature summary and our practical experiences in mass spectra interpretation, we provided some suggestions and strategies for manual peptide sequencing. This knowledge is important to improve the accuracy in protein and peptide sequence identification, especially for proteins originating from non-model organism without gene sequencing.In chapter 2, we investigate formation mechanism of diagnostic bn-44 peptide fragments of Dh HP-6 upon CID process. The singly charged precursor ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28(an)ions. High resolution mass spectra(HRMS) and isotope labeling experiments indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: 1. loss of CH3CHO(44.0262) from a Thr side chain; 2. loss of CO2(43.9898) from the oxazolone structure in C-terminus.In chapter 3, we investigate formation of cn ions in details. Low-energy CID remains the most popular and readily accessible activation method for protonated peptides which is characterized by amide bond cleavage and the formation of typical b/y ions.The observation of the c-type ion is relatively a rare event. Some previous research has highlighted that the formation of unusual cn ions upon CID process is related to the presence of special amino acids, and various mechanisms were proposed to help explain formation of these unusual fragments. To study the effect of N-terminal fixed charge, charge state, competing reaction and amino acid composition, experiments including HRMS, amino acid substitution, H/D exchange were performed. These results suggest that the unusual c ions are formed by Mc Lafferty-type rearrangement via charge-remote fragmentation, which is confirmed by the following DFT calculations and in accordance with the experimental results of previous research.In chapter 4, we investigate the formation of b2+H2O ions formed by DhHP-6.Formation of b+H2O type ion is initiated by nucleophilic attack of C-terminal carboxyl group on adjacent N-terminal carbonyl carbon in CID process. However,amidation of the C-terminus in Dh HP-6 switched off the particular dissociation pathway. Meanwhile, substitution of Thr by Gly inarguably indicated that Thr residue played a pivotal role on the generation of b2+H2O ions. Inspired by the present knowledge of N?O acyl shift in solution, we first introduce a novel rearrangement fragmentation pathway to produce bn+H2O ions for Thr containing peptides in gas-phase. N?O acyl shift resulting in generation of an ester intermediate is the first and most critical step, and then further fragmentation of the ester isomer leads to the formation of bn+H2O ions. On the basis of LC-MS/MS behavior of the methylated peptides, we find that amide N-methylation of Thr significantly promotes the N?O acyl shift both in solution and gas-phase. Density functional theory(DFT)calculations were used to elucidate the formation mechanism of this type ion in gas-phase.In chapter 5, we perform structural confirmation to two special peptide drugs by using mass spectrometric strategy. Although MS/MS has been routinely used for peptide sequencing, some specific or unknown PTMs and artificial chemical modification in peptides bring challenges. Isomerization of Asp is closely relating to protein degradation, diagnostic ions or ratio change in sequence ions were used to distinguish Asp isomers based on gas-phase fragmentation. In the commercial kit, quantitative and qualitative detection of isoaspartate is based on enzymatic reaction and HPLC detector. In this work, we can obtain the accurate number and position of isoaspartate through combination of enzymatic reaction and MS/MS analysis. Inspired by the strategy in proteomics, structure confirmation of peptide MAP-4 was performed. In the top-down analysis, complementary bn and [M-bn] ions were the major products although there are four branch chain; in the bottom-up analysis, enzymolysis products are in correspondence with theoretical products within high coverage of sequence ions.