We report on modifications made to a Paul-type quadrupole ion trap mass spectrometer and discuss its application in infrared ion spectroscopy experiments. Main modifications involve optical access to the trapped ions and hardware and software coupling to a variety of infrared laser sources at the FELIX infrared free electron laser laboratory. In comparison to previously described infrared ion spectroscopy experiments at the FELIX laboratory, we find significant improvements in efficiency and sensitivity. Effects of the trapping conditions of the ions on the IR multiple photon dissociation spectra are explored. Enhanced photo-dissociation is found at lower pressures in the ion trap. Spectra obtained under reduced pressure conditions are found to more closely mimic those obtained in the high-vacuum conditions of an Fourier transform ion cyclotron resonance mass spectrometer. A gas-mixing system is described enabling the controlled addition of a secondary gas into helium buffer gas flowing into the trap and allows for ion/molecule reactions in the trap. The electron transfer dissociation (ETD) option of the mass spectrometer allows for IR structure characterization of ETD-generated peptide dissociation products.

Glycosyl cations are crucial intermediates formed during enzymatic and chemical glycosylation. The intrinsic high reactivity and short life-time of these reaction intermediates makes them very challenging to characterize using spectroscopic techniques. Herein, we report the use of collision induced dissociation tandem mass spectrometry to generate glycosyl cations in the gas phase followed by infrared ion spectroscopy using the FELIX infrared free electron laser. The experimentally observed IR spectra were compared to DFT calculated spectra enabling the detailed structural elucidation of elusive glycosyl oxocarbenium and dioxolenium ions.

Tandem mass spectrometry occupies a principle place among modern analytical methods and drives many developments in the ‘omics’ sciences. Electron attachment induced dissociation methods, as alternatives for collision-induced dissociation have profoundly influenced the field of proteomics, enabling among others the top-down sequencing of entire proteins and the analysis of post-translational modifications. The technique, however, produces more complex mass spectra and its radical-driven reaction mechanisms remain incompletely understood. Here we demonstrate the facile structural characterization of electron transfer dissociation generated peptide fragments by infrared ion spectroscopy using the tunable free-electron laser FELIX, aiding the elucidation of the underlying dissociation mechanisms. We apply this method to verify and revise previously proposed product ion structures for an often studied model tryptic peptide, [AlaAlaHisAlaArg+2H]²⁺. Comparing experiment with theory reveals that structures that would be assigned using only theoretical thermodynamic considerations often do not correspond to the experimentally sampled species.

Small molecule identification is a continually expanding field of research and represents the core challenge in various areas of (bio)analytical science, including metabolomics. Here, we unequivocally differentiate enantiomeric N-acetylhexosamines in body fluids using infrared ion spectroscopy, providing orthogonal identification of molecular structure unavailable by standard liquid chromatography/high-resolution tandem mass spectrometry. These results illustrate the potential of infrared ion spectroscopy for the identification of small molecules from complex mixtures.

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients. Funding. Society for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).