Methodological advances for the measurement of the D-amino acids across the metazoa: From mollusks to man

Capillary Electrophoresis, Liquid Chromatography, Mass Spectrometry, Single Cell, Chiral Separations

Patel, Amit V

关键词: Capillary Electrophoresis, Liquid Chromatography, Mass Spectrometry, Single Cell, Chiral Separations;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/101139/PATEL-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The amino acids are a class of biomolecules which is obligatory to terrestrial life, alongside nucleic acids, fatty acids and monosaccharides. The amino acids, with the exception of glycine, are chiral molecules which exist as either L- or D-amino acids. Although identical with regard to physical and chemical properties, the enantiomeric forms rotate plane-polarized light in opposite directions. The difference in stereoconfiguration of D- and L-amino acids can impact their biochemical properties, as a consequence of differential interactions with other chiral biomolecules, often leading to substantial differences in physiological significance.Originally, the L-amino acids and D-sugars were considered to be isomeric forms naturally present in living systems, often referred to as the homochirality of life. Over time, in the wake of measurements of D-amino acids in bacteria and animals dating back to the mid-1900s, the idea of the homochirality of life was adapted to state that chiral biomolecules exist almost exclusively in one enantiomeric form. After several decades of research, it is now evident that the D-amino acids are found in every domain of life (Archaea, Bacteria and Eukarya). Beyond their ubiquitous presence, a few D-amino acids have been demonstrated to act as cell-to-cell transmitters and functional roles in the central nervous and endocrine systems have been uncovered.In spite of great progress made in our understanding of the D-amino acids in nature over the past several decades, many questions remain regarding the D-amino acids, the complement of proteins involved in their production, transduction, transport and degradation as well as functional importance. These gaps in knowledge emanate from the analytical challenges in D-amino acids measurement. Beyond the conventional challenges of bioanalytical chemistry, analyzing the D-amino acids requires approaches which can discriminate on the basis of chirality as well as overcome the challenges presented by heterogeneous distribution and low abundance. Herein described is a body of work relating to methodological advances to facilitate the analysis of the D-amino acids as well as the characterization of D-amino acids in cells and tissues from the central nervous and endocrine systems of animals, from mollusks to man. Regarding the goal of developing approaches for D-amino acid analysis, chiral separations involving capillary electrophoresis and liquid chromatography have been developed and characterized. Capillary electrophoresis paired with laser-induced fluorescence was leveraged for single cell analysis, and an online sample preconcentration method was developed for further enhancement of sensitivity. Liquid chromatography couple with mass spectrometry was used for the measurement of the D-amino acids as well as the execution of pulse-chase experiments. Additionally, protocols for the extraction, purification and derivatization of the D-amino acids have been explored. Together, these methodological advances were paired with experiment design and targeted sampling in order to characterize the D-amino acids in the central nervous and endocrine systems.