Metabolism of beta-endorphin in plasma studied by liquid chromatography-electrospray ionization mass spectrometry.
Degradation of synthetic human beta-endorphin by a human plasma proteinase was studied with high-performance liquid chromatography in combination with mass spectrometry. The peptide was metabolized at a rate of 25 pmol/min to the major fragments beta-endorphin (1-19) and (20-31), the latter reported as a potent inhibitor of morphine- and beta-endorphin-induced analgesia in mice. The proteinase responsible for this process was classified as a metal-dependent serine proteinase and was effectively inactivated by phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Identification of the products formed during the enzymatic reaction was performed by liquid chromatography on-line with electrospray mass spectrometry, using a reversed-phase or a novel size-exclusion column capable of separating molecules between 0.1-7 kilodaltons. Peptide sequences were verified by tandem mass spectrometry experiments. The conversion of beta-endorphin may have physiological implications in the mechanism of pain. The obtained data suggest that several precautions should be considered during recovery and measurement of beta-endorphin in plasma by immunological techniques. The applied strategy may also be useful for studying metabolism of various peptidergic compounds with potential pharmacological significance.
Sandin
J
J
Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden. johan.sandin@cmm.ki.se
Nylander
I
I
Silberring
J
J
D000328:Adult; D000595:Amino Acid Sequence; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000097:blood*; D001615:beta-Endorphin / Q000378:metabolism*
D010446:Peptide Fragments; C111754:endorphin (1-20); C111755:endorphin (20-31); D001615:beta-Endorphin; D012697:Serine Endopeptidases