Abstract
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.
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Abbreviations
- CID:
-
Collision-induced dissociation
- FWHM:
-
Full width at half maximum
- Glu-Glu-Phe:
-
Glutamyl-glutamyl-phenylalanine
- Glu-Phe:
-
Glutamyl-phenylalanine
- HCD:
-
High-energy collisional dissociation
- HMDB:
-
The human metabolome database
- HMGCLD:
-
3-Hydroxy-3-methylglutaryl-CoA lyase deficiency
- HRMS:
-
High-resolution mass spectrometry
- IEM:
-
Inborn error of metabolism
- LC:
-
Liquid chromatography
- MSI:
-
Metabolomics standards initiative
- MSn :
-
Multistage fragmentation mass spectrometry
- NMR:
-
Nuclear magnetic resonance
- PAHD:
-
Phenylalanine hydroxylase deficiency
- Phe:
-
Phenylalanine
- Phe-Phe:
-
Phenylalanyl-phenylalanine
- PKU:
-
Phenylketonuria
- ppm:
-
Parts per million
- QTOF MS:
-
Quadrupole time-of-flight mass spectrometry
- RDB:
-
Ring-double-bond
- RT:
-
Retention time
- Tyr:
-
Tyrosine
- UHPLC:
-
Ultra-high performance liquid chromatography
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Acknowledgments
This work was supported by NPU I (LO1304) and Czech Science Foundation Grant 15-34613 L. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.
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All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000.
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Informed consent was obtained from all patients for being included in the study.
This article does not contain any studies with animal subjects performed by the any of the authors.
Conflict of interest
J. Václavík, K. L. M. Coene, I. Vrobel, L. Najdekr, D. Friedecký, R. Karlíková, L. Mádrová, A. Petsalo, U. F. H. Engelke, A. van Wegberg, L. A. J. Kluijtmans, T. Adam and R. A. Wevers declare that they have no conflict of interest.
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Václavík, J., Coene, K.L.M., Vrobel, I. et al. Structural elucidation of novel biomarkers of known metabolic disorders based on multistage fragmentation mass spectra. J Inherit Metab Dis 41, 407–414 (2018). https://doi.org/10.1007/s10545-017-0109-4
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DOI: https://doi.org/10.1007/s10545-017-0109-4