Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of beta-endorphin by endothelial cells.
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 beta-endorphin (h beta E) 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 microliters/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 h beta E was found to be primarily attacked by the enzymes of the endothelial cells.
Brudel
M
M
Institute of Molecular Pharmacology, Berlin, Germany.
Kertscher
U
U
Schröder
D
D
Melzig
M F
MF
Mehlis
B
B
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D001011:Aorta; D002268:Carboxypeptidases / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D001615:beta-Endorphin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D001615:beta-Endorphin; D002268:Carboxypeptidases; D000626:Aminopeptidases