LC/MS evaluation of metabolism and membrane transport of bombesin peptides |
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Authors: | Dongyu Gu Ying Ma Gang Niu Yongjun Yan Lixin Lang Haji Akber Aisaand Haokao Gao Dale O Kiesewetter Xiaoyuan Chen |
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Institution: | (1) Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), 31 Center Dr, Suite 1C14, Bethesda, MD 20892-2281, USA;(2) Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, China;(3) Graduate University of the Chinese Academy of Sciences, Beijing, China;(4) NIBIB/NIH, 10 Center Drive MSC 1180, Bethesda, MD 20892, USA; |
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Abstract: | Two bombsin peptides, GRPR agonist Aca-QWAVGHLM-NH2] 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. |
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