On-Tissue Chemical Derivatization of Catecholamines Using 4-(N-Methyl)pyridinium Boronic Acid for ToF-SIMS and LDI-ToF Mass Spectrometry Imaging

Abstract

The analysis of small polar compounds with ToF-SIMS and MALDI-ToF-MS have been generally hindered by low detection sensitivity, poor ionization efficiency, ion suppression, analyte in-source fragmentation, and background spectral interferences from either a MALDI matrix and/or endogenous tissue components. Chemical derivatization has been a well-established strategy for improved mass spectrometric detection of many small molecular weight endogenous compounds in tissues. Here, we present a devised strategy to selectively derivatize and sensitively detect catecholamines with both secondary ion ejection and laser desorption ionization strategies, which are used in many imaging mass spectrometry (IMS) experiments. Chemical derivatization of catecholamines was performed by a reaction with a synthesized permanent pyridinium-cation-containing boronic acid molecule, 4-(N-methyl)pyridinium boronic acid, through boronate ester formation (boronic acid−diol reaction). The derivatization facilitates their sensitive detection with ToF-SIMS and LDI-ToF mass spectrometric techniques. 4-(N-Methyl)pyridinium boronic acid worked as a reactive matrix for catecholamines with LDI and improved the sensitivity of detection for both SIMS and LDI, while the isotopic abundances of the boron atom reflect a unique isotopic pattern for derivatized catecholamines in MS analysis. Finally, the devised strategy was applied, as a proof of concept, for on-tissue chemical derivatization and GCIB-ToF-SIMS (down to 3 μm per pixel spatial resolution) and LDI-ToF mass spectrometry imaging of dopamine, epinephrine, and norepinephrine in porcine adrenal gland tissue sections. MS/MS using collision-induced dissociation (CID)-ToF-ToF-SIMS was subsequently employed on the same tissue sections after SIMS and LDI mass spectrometry imaging experiments, which provided tandem MS information for the validation of the derivatized catecholamines in situ. This methodology can be a powerful approach for the selective and sensitive ionization/detection and spatial localization of diol-containing molecules such as aminols, vic-diols, saccharides, and glycans along with catecholamines in tissue sections with both SIMS and LDI/MALDI-MS techniques.

In situ Metabolite Mass Spectrometry Imaging: New Insights into the Adrenal Gland

“…Particularly, MS 3 was necessary to produce the specific fragments to enable visualization of derivatized norepinephrine. A more accurate localization of dopamine, epinephrine and norepinephrine in the modular region also was achieved in another study [51]. By applying 4-(N-methyl)pyridiniumboronic acid [4-(NMe)PyB(OH) 2 ] derivatization, both LDI-TOF-MSI and gas cluster ion beam (GCIB)-TOF-SIMS analysis yielded a similar characteristic spatial distribution of epinephrine, norepinephrine and dopamine in porcine adrenal gland tissue sections; these compounds localize in the peripheral, central and entire medulla region, respectively, as shown in ▶ Fig.…”

“…Although high-resolution mass analyzers such as FTICR-MS (i. e., 100 000 mass resolution for common tissue metabolites) allow relatively accurate and confident assignments of ion peaks to chem- ical formulas, a lack of detection sensitivity can hinder its application to the molecular imaging due to low ionization efficiency, ion suppression by abundant species, and background spectral interference from either the matrix and/or tissue components [51,52]. Low sensitivity in the adrenal gland MSI study is particularly prominent for neutral steroids as they have relatively low abundance, poor ionization yields (i. e., lack of hydrogen donor or acceptor moieties), and susceptibility to ion suppression of lipids and proteins [53].…”

“…These findings have led to deeper insight into the metabolites and hormone adaptation in the functional and dysfunctional adrenal gland and adrenal tumors as will be shown in later cases. Specific to the adrenal gland, a few studies also have focused on the improvement of the detection and localization of adrenal-specific hormones, including for catecholamines [50][51][52] and particularly for poorly ionized and isomeric steroids, leading to new hypothesis of adrenal disease pathogenesis, as will be reported later in this review [49,52,53].…”

Detection of a High-Turnover Serotonin Circuit in the Mouse Brain Using Mass Spectrometry Imaging

“…However, it cannot discriminate between intracellular compartments (i.e., soma, dendrites, and axons) and intracellular/extracellular monoamines, since the minimum spatial resolution for the monoamines is about 1–3 μm (Kaya et al., 2018, Kompauer et al., 2016, Passarelli et al., 2017). Although a higher spatial resolution <1 μm may be achieved using secondary ion mass spectrometry in the near future (Kaya et al., 2018, Passarelli et al., 2017), the use of MSI combined with other techniques that detect extracellular monoamines (i.e., microdialysis [Chen et al., 1999], fast-scan cyclic voltammetry [Borue et al., 2010]) as well as optogenetics (Takata et al., 2018, Watanabe et al, 2018) or fiber photometry (Tsutsui-Kimura et al., 2017) is essential to better understand the monoaminergic regulation of animal behaviors. We anticipate that further MSI analysis of monoamine turnover will provide direct evidence of synthesis, transport, and regional availability of the brain monoamines.…”

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI

“…After rapid derivatization reaction at room temperature and spraying of CHCA as the MALDI matrix, positively charged N-methylpyridinium derivate contributed to improved ionization efficiency of several cannabinoids and their metabolites including Δ 9 -tetrahydrocannabinol (THC) in hair, which were soaked in solutions with the respective analytes. Furthermore, THC could be visualized with FMPTS derivatization in hairs from a known cannabis consumer.Derivatization of diol in catechol with (N-Me)Py + B(OH) 2 Kaya and coworkers[79] synthesized 4-(N-methyl)pyridinium boronic acid ((N-Me)Py + B(OH) 2 ), which was applied for derivatization of catecholamines including dopamine, epinephrine, and norepinephrine in porcine adrenal gland tissue sections. Boronic acid derivative can readily react with the diol moiety of catechol (ortho isomer of dihydroxybenzene), forming five-membered boronate esters.…”