METABOLOMICS Structural elucidation of novel biomarkers of known metabolic disorders based on multistage fragmentation mass spectra Jan Václavík 1,2 & Karlien L. M. Coene 3 & Ivo Vrobel 1,2 & Lukáš Najdekr 1,2 & David Friedecký 1,2 & Radana Karlíková 1,2 & Lucie Mádrová 1,2 & Aleksanteri Petsalo 1 & Udo F. H. Engelke 3 & Annemiek van Wegberg 4 & Leo A. J. Kluijtmans 3 & Tomáš Adam 1,2 & Ron A. Wevers 3 Received: 28 July 2017 /Revised: 2 October 2017 /Accepted: 22 October 2017 # SSIEM 2017 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 po- tential novel diagnostic biomarkers. Current separation tech- niques coupled to high-resolution mass spectrometry provide a powerful tool for structural elucidation of unknown com- pounds in complex biological matrices. This is a proof-of- concept study testing this methodology to determine the mo- lecular 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 molecu- lar structures, spectral databases were searched and fragmen- tation 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 phenylke- tonuria 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 defi- ciency, three positional isomers of 3-methylglutaconyl carni- tine could be detected in patient plasma. Our results highlight the applicability of current accurate mass multistage fragmen- tation techniques for structural elucidation of unknown metab- olites in human biofluids, offering an unprecedented opportu- nity to gain further biochemical insights in known inborn er- rors of metabolism by enabling high confidence identification of novel biomarkers. Keywords Next generation metabolic screening . Metabolomics . Structural elucidation . Biomarkers . Phenylketonuria . 3-hydroxy-3-methylglutaryl coenzyme a lyase deficiency Introduction The diagnosis and monitoring of patients suffering from in- born errors of metabolism (IEMs) traditionally relies on targeted metabolite analyses of human biofluids. Recently, the use of untargeted metabolomic techniques like proton nu- clear magnetic resonance (NMR) spectroscopy and high- resolution mass spectrometry has found its way into the Jan Václavík, Karlien L. M. Coene, Tomáš Adam and Ron A. Wevers contributed equally to this work. Communicated by: Nenad Blau Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10545-017-0109-4) contains supplementary material, which is available to authorized users. * Tomáš Adam tomas.adam@fnol.cz 1 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 2 Laboratory of Inherited Metabolic Disorders, Department of Clinical Chemistry, University Hospital in Olomouc, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic 3 Translational Metabolic Laboratory - 830 TML, Department of Laboratory Medicine, Radboud University Medical Centre, Geert Grooteplein 10, 6525, GA Nijmegen, the Netherlands 4 Department of Gastroenterology, Radboud University Medical Centre, Geert Grooteplein 10, 6525, GA Nijmegen, the Netherlands J Inherit Metab Dis https://doi.org/10.1007/s10545-017-0109-4