Identification of PTH-Amino Acids by HPLC

Abstract

Protein sequence analysis employing Edman degradation chemistry commonly uses high-performance liquid chromatography (HPLC) separation as the means for identification of the PTH-amino acid (phenylthiohydantoin amino acid) produced at each cycle. In fact, all modern automated protein sequencing instruments come with “on-line” HPLC detection as standard equipment. This method of identification of PTH-amino acids is dependent on essentially two factors: resolution of one PTH-amino acid from another and coincidence of elution position of the unknown PTH-amino acid with that of a known standard. Because the identification of the PTH-amino acid is based almost solely on its elution position, the ideal situation is that each PTH-amino acid elute at a unique position in the chromatogram. This is accomplished by the choice of column, mobile phase, and elution program. However, even if conditions are found to separate every PTH-amino acid from one another, identification still depends on matching the elution position with a standard of known structure. The method is straightforward for the genetically coded amino acids but becomes more problematic for modified amino acids that are not routinely encountered. Because the method does not provide a direct identification of the PTH-amino acid, additional analysis by chemical or physical means may often be necessary to provide unequivocal identification of nonstandard PTH-amino acids. However, it is often helpful to have some knowledge of where known modified PTH-amino acids elute in these systems.