The Application Of Mass Spectrometry In Chiral Amino Acid Analysis

Chirality is the common phenomenon and one of the most important basic characters in nature, which is reflected by chiral molecules, such as amino acids, nucleotides and monosaccharids and so on. Slightly changing the configuration of amino acid in protein will affect the whole sequence of the protein, further causing some related diseases. By now, the existed method using mass spectrometry to do chiral analysis still relies on the comparation of standard, inducing chiral environment which is hard to meet the requirement of fast analysis, especially for the mixture analysis and biological system analysis. By considering the limitations of previous methods in MS chiral analysis and the requirement for high-throughput analysis, in this thesis, we selected the common chiral amino acids and peptides as the research targets. We successfully applied the MS method to fast analyze their absolute configuration both qualitatively and quantitatively without comparing with standards, especially making a breakthrough in mixture analysis and biological system analysis. The detail studies are shown in the followings:1. In-source aldolization reaction in electrospray ionization mass spectrometry. Unexpected [M+15]+ ions were formed during the analysis of aromatic aldehydes by use of methanol in positive-ion electrospray ionization mass spectrometry. Deuterium-labeling experiment, high performance liquid chromatography-MS experiment, theoretical calculations were performed to identify the formation of [M+15]+ ion. The proposed reaction mechanism is a gas-phase aldol reaction between protonated aromatic aldehydes and methanol via proton-transport catalysis. This unique gas-phase ion/molecule reaction offers a novel approach for direct identification of aromatic aldehyde and provides new ideas for developing an in-situ chiral analysis method.2. Qualitative and quantitative analysis of enantiomers by mass spectrometry:the application of a simple chiral chloride probe via rapid in-situ reaction. The method is based on a chemical derivation process that uses a simple reagent, L-1-(phenylsulfonyl) pyrrolidine-carbonyl chloride, as the probe. The ratio of amino acid spiked at different concentrations (enantiomeric excess) in both organic solution and dog plasma is determined by establishing calibration curves. The present method is demonstrated as a new and practical technique of rapidly and sensitively determining enantiomers of amino compounds.3. Facile assignment of the configuration of amino acid residues in peptides using mass spectrometry. A pair of novel isotopic labeling mass tagged chiral derivatives were designed and synthesized. These agents can label amino acids in-situ. The configurations of amino acids and peptide residues can be determined by analyzing the mass spectra of the imine product. Both NMR analysis and theoretical calculations verified that the chiral recognition was caused by a kinetic difference between the analytes and reagents, that was strongly dependent on hydrogen bond interaction, π-π stacking and steric interactions. This assay is suitable for mixture analysis without comparison with standards and can be applied for the high-throughput recognition of the configuration of amino acid residues in peptides.4. Differentiation peptide isomers based on charge remote fragmentation. The analysis of tripeptide, pentapeptide and heptapeptide coupled with their isomers was performed using ESI-MS/MS. The major fragments observed in the protonated peptides were mainly those expected ions from the b and y series. Other ions from neutral loss of water or ions due to cyclization reaction were detected. It’s hard to study the residues configuration on the fragmentation pattern of all peptide isomers from such complicated spectra. However, when induced Pd (Ⅱ) ions into the solution, the representative MS/MS spectrum for [peptide+Pd2+-H+]+ ion reveals a series of b-type ions ranging from b2 to b4 and the abundances of these b ions were different which is useful for determining the configuration. A charge remote fragmentation pathway was put forward to explain this phenomenon. Further researches based on NMR technique, deuterium experiment and molecular dynamics simulations strongly support the mechanism.5. The host-guest interaction of cyclofructans and their application in chiral amino acids anslysis. Cyclofructans is a new class of cyclic oligosaccharide hosts. Due to the special crown ether skeleton, cyclofructans gives different chemical activities compared with cyclodextrin. But the researches for detecting cyclofructans using mass spectrometry are still limited. The noncovalent complex interactions between cyclofructans and various amino acids and their binding constants have been characterized by means of mass spectrometry, nuclear magnetic resonance and theory calculations. Spectrophotometic studies demonstrated that their binding properties could be actuated by modulating the pH of the solution. Based on the researches of interaction mechanism, a new host-guest in-situ reaction method was developed for determining the chirality of aromatic amino acids.