Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of β-endorphin by endothelial cells

Abstract

An on-line HPLC-mass spectrometric procedure with an electrospray atmospheric pressure ionization (ESI-API) ion source was developed to identify the enzymatic degradation products (peptides) generated by incubation of human β-endorphin (hβE) with cultured aortic endothelial cells. The samples from the complex incubation mixture were prepurified and enriched using a small reversed-phase (RP) perfusion precolumn. Flow switching was applied to transfer the peptides from this precolumn to the analytical RP column of 2 or 0.32 mm I.D. and to separate them by gradient elution. The peptides were detected by means of an on-line coupled triple quadrupole mass spectrometer (TSQ 700) with an ESI-API ion source operated in the positive ion mode. This MS system behaves as a concentration sensitive detector at flow-rates from 5 to 150 μl/min. MS-MS experiments supported the unambiguous assignment of the peptide structures. Thus most of the peptide fractions were identified and the region 16-17-18 (-L-F-K-) of hβE was found to be primarily attacked by the enzymes of the endothelial cells.