Guan Fuyu, Fay Savannah, Li Xiaoqing, You Youwen, Robinson Mary A
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.
Drug Test Anal. 2020 Jun;12(6):771-784. doi: 10.1002/dta.2781. Epub 2020 Mar 24.
Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides were detected using their theoretical masses generated in silico, their appearance was monitored over the time course of incubation, and their identity was verified by their product ion spectra. Catabolites identified for chemotactic peptide, DALDA, dmtDALDA, deltorphins I and II, Hyp -dermorphin, Lys -dermorphin, and dermorphin analog are novel. A d-amino acid residue at position 2 or 1 of a peptide or next to its C-terminus protected the relevant terminal from degradation by exopeptidases, but such a residue at position 3 did not. A pGlu residue or N-methylation at the N-terminus of a peptide did not protect its N-terminal. Ethylamide at the C-terminus of a peptide provided the C-terminal protection from attacks by carboxypeptidases. The C-terminal Lys amide in DALDA, dmtDALDA, and Lys -dermorphin was susceptible to cleavage by plasma enzymes, which is the first report, to the authors' knowledge. The results from the present study provide insights into the stability of peptides in plasma.
生物活性肽对体育赛事的公正性构成了巨大威胁。检测这些肽对于在体育赛事中实施对它们的禁用至关重要。识别这些肽在血浆中离体形成的代谢产物可能会提高对它们的检测能力。在本研究中，使用亲水相互作用色谱（HILIC）与高分辨质谱（HRMS）联用，在不同孵育条件下检测了27种两端均有保护基团的生物活性肽在马血浆中的稳定性。在这27种肽中，13种在37°C孵育72小时后是稳定的，但其余14种稳定性较差。利用计算机模拟生成的理论质量检测了这14种肽的离体代谢产物，在孵育过程中监测它们的出现情况，并通过其产物离子光谱验证其身份。为趋化肽、DALDA、dmtDALDA、强啡肽I和II、Hyp-强啡肽、Lys-强啡肽和强啡肽类似物鉴定出的代谢产物是新的。肽的第2位或第1位或其C末端旁边的d-氨基酸残基可保护相关末端不被外肽酶降解，但第3位的此类残基则不能。肽的N末端的焦谷氨酸残基或N-甲基化并不能保护其N末端。肽的C末端的乙酰胺可保护C末端免受羧肽酶的攻击。据作者所知，DALDA、dmtDALDA和Lys-强啡肽中的C末端赖氨酸酰胺易被血浆酶切割，这是首次报道。本研究结果为肽在血浆中的稳定性提供了见解。
