Han Jie, Yang Qinyan, Zhi Zheng, Li Na, Wu Jian-Lin
State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau, 999078, China.
Liver Transplantation Center and HBP Surgery, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
Talanta. 2025 Apr 1;285:127345. doi: 10.1016/j.talanta.2024.127345. Epub 2024 Dec 6.
Carboxyl or carbonyl-containing metabolites (CoCCMs) are widely distributed in biological samples. Global profiling of CoCCMs is essential for ascertaining specific functions of metabolites and their potential physiological roles in biogenic activities. However, simultaneous determination of these compounds is hampered by poor ionization efficiency, vast polarity differences, wide discrepancy of concentration ranges. Herein, a novel bromine isotope derivatization reagent 5-bromo-2- hydrazinopyridine was employed for CoCCMs profiling by liquid chromatography-mass spectrometry (LC-MS). This method enabled rapid derivatization of 44 CoCCMs under mild conditions. Enhanced separation efficiencies, detection sensitivities, and distinctive MS fragmentation characteristics were observed. Furthermore, this method was demonstrated to be efficient in revealing metabolic alternations, and abnormal serum levels of 6-keto-PGF1α, 12(S)-HHTrE, 15(S)-HEPE and N-acetyl tryptophan were disclosed for the first time in Mycoplasma pneumoniae (MP) infectious patients. Finally, based on the distinctive 2 Da differences of molecular ion peak pairs with almost 1:1 intensity ratio originated from Br and Br isotopes, an MS-DIAL and Python assisted MS1 isotope screening-MS2 fragments characterization combination strategy was developed for rapid screening, classification, and identification of detected CoCCMs. A total of 1069 CoCCMs were detected, of which 198 CoCCMs were identified in untargeted analysis. Statistical analysis revealed altered metabolic pathways, while glutamic acid, oxoglutaric acid, succinic acid, pyruvic acid, glyceric acid, and glycine were selected as potential biomarkers of MP infection. This bromine signature coded derivatization-LC-MS approach was proved to be a valuable tool for global probing of CoCCMs in biological samples with high sensitivity and broad coverage.
含羧基或羰基的代谢物（CoCCMs）广泛分布于生物样品中。对CoCCMs进行全面分析对于确定代谢物的特定功能及其在生物活性中的潜在生理作用至关重要。然而，这些化合物的同时测定受到电离效率低、极性差异大、浓度范围差异大的阻碍。在此，一种新型溴同位素衍生试剂5-溴-2-肼基吡啶被用于通过液相色谱-质谱联用（LC-MS）对CoCCMs进行分析。该方法能够在温和条件下对44种CoCCMs进行快速衍生化。观察到分离效率、检测灵敏度提高，以及独特的质谱碎片特征。此外，该方法被证明在揭示代谢变化方面是有效的，并且首次在肺炎支原体（MP）感染患者中发现了6-酮-PGF1α、12（S）-HHTrE、15（S）-HEPE和N-乙酰色氨酸的血清水平异常。最后，基于源自Br和Br同位素的分子离子峰对具有几乎1:1强度比的独特2 Da差异，开发了一种MS-DIAL和Python辅助的MS1同位素筛选-MS2碎片表征组合策略，用于快速筛选、分类和鉴定检测到的CoCCMs。总共检测到1069种CoCCMs，其中198种CoCCMs在非靶向分析中被鉴定。统计分析揭示了代谢途径的改变，同时选择谷氨酸、氧代戊二酸、琥珀酸、丙酮酸、甘油酸和甘氨酸作为MP感染的潜在生物标志物。这种溴标记编码衍生化-LC-MS方法被证明是一种用于高灵敏度和广泛覆盖地全面探测生物样品中CoCCMs的有价值工具。
