Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital in Olomouc, Hněvotínská 5, 775 15, Olomouc, Czech Republic.
Laboratory of Inherited Metabolic Disorders, Department of Clinical Chemistry, University Hospital in Olomouc, I.P. Pavlova 6, 775 20, Olomouc, Czech Republic.
J Inherit Metab Dis. 2018 May;41(3):407-414. doi: 10.1007/s10545-017-0109-4. Epub 2017 Nov 14.
Specific diagnostic markers are the key to effective diagnosis and treatment of inborn errors of metabolism (IEM). Untargeted metabolomics allows for the identification of potential novel diagnostic biomarkers. Current separation techniques coupled to high-resolution mass spectrometry provide a powerful tool for structural elucidation of unknown compounds in complex biological matrices. This is a proof-of-concept study testing this methodology to determine the molecular structure of as yet uncharacterized m/z signals that were significantly increased in plasma samples from patients with phenylketonuria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. A hybrid linear ion trap-orbitrap high resolution mass spectrometer, capable of multistage fragmentation, was used to acquire accurate masses and product ion spectra of the uncharacterized m/z signals. In order to determine the molecular structures, spectral databases were searched and fragmentation prediction software was used. This approach enabled structural elucidation of novel compounds potentially useful as biomarkers in diagnostics and follow-up of IEM patients. Two new conjugates, glutamyl-glutamyl-phenylalanine and phenylalanine-hexose, were identified in plasma of phenylketonuria patients. These novel markers showed high inter-patient variation and did not correlate to phenylalanine levels, illustrating their potential added value for follow-up. As novel biomarkers for 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, three positional isomers of 3-methylglutaconyl carnitine could be detected in patient plasma. Our results highlight the applicability of current accurate mass multistage fragmentation techniques for structural elucidation of unknown metabolites in human biofluids, offering an unprecedented opportunity to gain further biochemical insights in known inborn errors of metabolism by enabling high confidence identification of novel biomarkers.
特定的诊断标志物是有效诊断和治疗先天性代谢缺陷（IEM）的关键。非靶向代谢组学可用于鉴定潜在的新型诊断生物标志物。当前的分离技术与高分辨率质谱相结合，为复杂生物基质中未知化合物的结构阐明提供了强大的工具。这是一项概念验证研究，旨在测试该方法，以确定在苯丙酮尿症和 3-羟基-3-甲基戊二酰辅酶 A 裂解酶缺乏症患者的血浆样本中显著增加的尚未表征的 m/z 信号的分子结构。使用能够进行多级碎裂的混合线性离子阱-轨道阱高分辨率质谱仪，获取未表征的 m/z 信号的精确质量和产物离子谱。为了确定分子结构，搜索了光谱数据库并使用了碎裂预测软件。这种方法能够阐明潜在用作 IEM 患者诊断和随访生物标志物的新型化合物的结构。在苯丙酮尿症患者的血浆中鉴定出两种新的缀合物，即谷氨酰-谷氨酰-苯丙氨酸和苯丙氨酸-己糖。这些新型标志物在患者间表现出高度的变异性，与苯丙氨酸水平无关，表明它们在随访方面具有潜在的附加价值。作为 3-羟基-3-甲基戊二酰辅酶 A 裂解酶缺乏症的新型生物标志物，可以在患者血浆中检测到 3-甲基戊烯二酰肉碱的三个位置异构体。我们的结果强调了当前准确质量多级碎裂技术在人类生物流体中未知代谢物结构阐明中的适用性，通过能够高置信度地鉴定新型生物标志物，为已知先天性代谢缺陷提供了进一步生化见解的前所未有的机会。
