LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY OF 4-(3-PYRIDINYLMETHYLAMINOCARBOXYPROPYL) PHENYLTHIOHYDANTOINS

We report the separation of 4-(3-pyridinylmethylaminocarboxypropyl) ph enylthiohydantoins by microbore reverse-phase high-performance liquid chromatography and their detection by on-line electrospray ionization mass spectrometry. These compounds are the products of the chemical st epwise degradation of polypeptides using 4-(3-pyridinylmethylaminocarb oxypropyl) phenyl isothiocyanate. We describe chromatographic conditio ns for on-column concentration of the analytes and for baseline separa tion of the isobaric amino acid derivatives of leucine and isoleucine. A commercially available protein sequencer was readily interfaced wit h the described analytical system and used for adsorptive sequence ana lysis of a panel of synthetic peptides containing collectively all 20 naturally occurring amino acids. On-line mass analysis of derivatives generated by automated sequencing confirmed that the derivatives were of the predicted mass and were detectable at comparable signal strengt h and sensitivity. Finally, we demonstrate that the additional selecti vity in data interpretation provided by mass analysis dramatically imp roves the signal-to-noise ratio and therefore enhances the ability to conclusively interpret protein and peptide sequence data. (C) 1995 Aca demic Press,Inc.