Ultra Performance Liquid Chromatography Analysis of Proteins and Synthetic Intermediates

The goal of this project was to develop standard operating procedures for analyzing proteins and synthetic intermediates using Ultra Performance Liquid Chromatography (UPLC), an experimental tool that is designed to separate complex mixtures into their individual components. Specifically, two classes of molecules were subjected to UPLC analysis: 1) proteins and 2) intermediates in the synthesis of fluorescent nonnatural amino acids. Proteins were analyzed following tryptic digest (i.e, after breaking the protein into well-defined fragments by the action of the digestive enzyme trypsin) using mass spectroscopy (MS) detection. In many cases, the results allow the definitive identification of the parent protein. The analysis of synthetic intermediates was focused on the synthesis of nonnatural amino acids. Three types of detection were used in the analysis of synthetic intermediates from the preparation of non-natural fluorescent amino acids: ultraviolet-visible (UV-vis), mass spectroscopy (MS), and fluorescence. The UPLC results provided information that complemented results of nuclear magnetic resonance (NMR) spectroscopy analysis. The combined UPLC and NMR results allowed the identification of intermediates and side products