title: LC/MS evaluation of metabolism and membrane transport of bombesin peptides.
abstract: Two bombsin peptides, GRPR agonist [Aca-QWAVGHLM-NH(2)] and antagonist [fQWAVGHL-NHEthyl] were evaluated. We employed the highly sensitive Waters Q-Tof Premier MS coupled with a UPLC system to identify the metabolites produced by rat hepatocytes or PC-3 human prostate cancer cells; and we utilized the AB/MDS 4000 Q-Trap LC/MS/MS system with highly sensitive quantitative and qualitative performance, to quantitatively analyze the internalization of GRPR agonist and antagonist in PC-3 cells. The major metabolites of both GRPR agonist and antagonist were the result of peptide bond hydrolysis between W and A which was demonstrated by observation of the N-terminal fragment m/z 446 (Aca-QW-OH) for agonist and m/z 480 (fQW-OH) for antagonist. Both peptides were also hydrolyzed between A and V which formed peaks m/z 517 [Aca-QWA-OH] and m/z 555 (VGHLM-NH2) for the agonist and m/z 551 [fQWA-OH] and m/z 452 (VGHL-NHEthyl) for the antagonist. The peptide agonist also formed a unique metabolite that resulted from hydrolysis of the C-terminal amide. The antagonist showed significantly slower metabolism as compared to the agonist in both rat hepatocytes and PC-3 cells. The antagonist also showed significantly lower PC-3 cell internalization rate than that of the agonist. In conclusion, the metabolism profiles of both GRPR agonist and antagonist peptides were identified by LC/MS. The antagonist peptide was more stable than the agonist peptide in rat hepatocyte incubation. One major factor could be the hydrolysis-resistant C-terminal L-NHEthyl group compared with the unsubstituted amide of the agonist. Another factor could be different amino acid sequences of the agonist and antagonist that may also influence the enzymatic hydrolysis. The antagonist ligand is potentially more useful for receptor-targeted imaging due primarily to its higher metabolic stability.
authors.affiliation: Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 31 Center Dr, Suite 1C14, Bethesda, MD, 20892-2281, USA.
authors.forename: Dongyu
authors.initials: D
authors.lastname: Gu
authors.forename: Ying
authors.initials: Y
authors.lastname: Ma
authors.forename: Gang
authors.initials: G
authors.lastname: Niu
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Yan
authors.forename: Lixin
authors.initials: L
authors.lastname: Lang
authors.forename: Haji Akber
authors.initials: HA
authors.lastname: Aisa
authors.forename: Haji Akber
authors.initials: HA
authors.lastname: Aisaand
authors.forename: Haokao
authors.initials: H
authors.lastname: Gao
authors.forename: Dale O
authors.initials: DO
authors.lastname: Kiesewetter
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D001692:Biological Transport; D001839:Bombesin / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018004:Receptors, Bombesin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D010455:Peptides; D018004:Receptors, Bombesin; D001839:Bombesin