ADVANCES IN LIQUID CHROMATOGRAPHY AND LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY FOR THE CHIRAL ANALYSIS OF AMINO ACIDS AND DIFFERENTIATION OF ISOMERIC AMINO ACID RESIDUES IN PEPTIDES/PROTEINS FROM COMPLEX MATRICES
Abstract
Amino acids are essential building blocks in all living organisms. Initially it was believed that only L-amino acids were relevant in higher organisms, and D-amino acids were laboratory artifacts. Today, various D-amino acids have been detected in different organisms, including humans, and some even play essential roles in biological processes. In addition, abnormal D-amino acid levels have been reported in patients with different diseases. The variations in D-amino acid levels might be of some diagnostic value. However, our knowledge of D-amino acids remains limited and most of the D-amino acids are not well investigated due to the lack of a comprehensive analytical platform. Analysis of D-amino acids in biological samples is challenging, because of the interference of large amounts of L-amino acids and a plethora of other indigenous compounds. The complete analysis of all D-amino acids requires a long period of time. Therefore, the goals of this dissertation are to 1) enhance current analytical methods for analyzing D-amino acids in biological samples, and 2) provide information that further studies can reference in finding functions related to pathology and potentially other unexplored D-amino acids processes.
Novel methodologies, based on high performance liquid chromatography-paired ion electrospray ionization mass spectrometry (HPLC-PIESI-MS), two dimension-HPLC (2D-HPLC), and HPLC-tandem mass spectrometry (HPLC-MS/MS), were developed and evaluated for the analysis of free L- and D-amino acids and peptides-containing isomeric amino acid residues from complex matrices. For the achiral analysis of amino acids, an ultrasensitive detection method using PIESI-MS was developed for the analysis of Fmoc-amino acids in urine samples. This method showed improved detection sensitivity down to sub-pg level. A 2D-HPLC chiral separation method with high sensitivity and selectivity was developed for the comprehensive baseline study of L- and D-amino acids in mouse brain and blood. A simple, rapid and sensitive chiral analysis method was developed using HPLC-MS/MS for the simultaneous analysis of 20 common amino acids and their enantiomeric compositions in wild-type mice and mutant mice lacking D-amino acid oxidase activity. Also, the intracellular and extracellular profiles of L- and D-amino acids in human breast cancer cells (MCF-7) and non-tumorigenic epithelial breast cells (MCF-10A) were reported for the first time using this HPLC-MS/MS method. We have also developed the first comprehensive analytical platform that can separate all 20 possible β-amyloid peptides containing Asp, isoAsp, and Ser isomers. Using this simple and high-throughput separation method, we will be able to identify and quantify Asp, isoAsp, and Ser isomers at every position of the β-amyloid peptides from Alzheimer’s patients simultaneously.