Use of narrow-bore high-performance liquid chromatography for microanalysis of protein structure

Abstract

We report here systematic approaches to microanalysis of protein structures using narrow-bone high-performance liquid chromatography of protein, peptide and derivatized [phenylthiocarbamyl (PTC-) and phenylthiohydantoinyl (PTH-)] amino acids. The utilization of columns with small diameters (2 mm or less) has improved resolution and sensitivity and enabled protein structure analysis at the low pmol level. Preparative isolation of proteins and peptides of pmol quantity is achieved and separation and identification of PTC- and PTH-amino acids can be routinized at the low pmol to subpmol level. The use of diode-array detection enables simultaneous multiple-wavelength monitoring and spectral retreat, which greatly enhances flexibility and usefulness of the present methods. In combination with protein microsequencing techniques, these narrow-bore high-performance liquid chromatographic procedures can facilitate structural analysis of minutely available proteins of biochemical and physiological significance.