DERIVATIZATION STRATEGIES FOR SENSITIVE MONITORING OF SMALL MOLECULES ON TISSUE SAMPLES

   pEČINKA L.^1,2, MORÁŇ l.^3,4, BŘEZINA A.^ 1,5, VESSELÁ T.^ 3, GABRIELOVÁ V.^ 3, HAVEL J.^1,2,
                      MARCHETTI-DESCHMANN M.^6, HAMPL A.^2,3, VAŇHARA P.^2,3*

                 ^1Faculty of Science, Masaryk University, Brno, Czech Republic,^

    ^2International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech
                                             Republic,

                 ^3Faculty of Medicine, Masaryk University, Brno, Czech Republic,

^4Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Brno,
                                          Czech Republic,

          ^5RECETOX Centre, Faculty of Science, Masaryk University, Brno, Czech Republic

   ^6Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna,
                                             Austria.

                                    e-mail: 436922@mail.muni.cz

Study of small molecules (mostly known as metabolites) for biomarker discovery consists of a
complete set of metabolites in complex biological systems (databases contain ~ 115 000
metabolites). Metabolome is much smaller than proteome (1.8 million different proteins) making it
relatively simple for data analysis. On the other hand, metabolites are chemically diverse
compounds that occur at vide range of concentrations (ten orders of magnitude) which makes
metabolomics analysis challenging.

Gas/Liquid Chromatography-Mass Spectrometry (GC/LC-MS) has become the gold standard for metabolome
analyses of clinical samples. However, these approaches require tissue homogenization prior to
analysis and they are only hardly able to provide information concerning the spatial localization
molecules of interest. MALDI MSI offers a label-free, unbiased visualization of the spatial
distributions of biomolecules without almost any sample preparation prior to analysis. This method
easily correlates with different histopathological features of the tumor tissue, allowing further
insights into the tumor environment.

Various chemical derivatization procedures have been extensively studied to enhance ionization
efficacy, modify retention time, and shift molecular mass to a higher value. Functional groups
including aldehyde, primary amines, carboxylic acid, and hydroxyl group have been targeted using
derivatization strategies. Derivatization process can differ regarding sample types: in-solutions
derivatization for the MS homogenous biological solutions (cell/tissue extract, plasma, and other
biological fluids) or on-tissue derivatization concerning the spatial localization of biological
markers - Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI).

Herein, the method for the on-tissue derivatization of various small molecules has been tested and
optimized for the salivary gland tissue. Three derivatization reagents have been used: Girard
reagent T (GirT) – carbonyl group, coniferyl aldehyde (CA) – primary amine group, and
2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP-TS) – hydroxy group. Spatial distribution of
derivatized/non-derivatized molecules detected in MALDI-MSI has been visualized using commercial
software as well as using in-house developed script in R and Python programming language. Mass
spectra processing and exploratory analysis using commercial software were compared with in-house
developed pipelines. MALDI-MSI spatial segmentation maps were compared with the histopathological
annotation. These results underline the potential of on-tissue derivatization MALDI-MSI for
sensitive detection of metabolite’s spatial distribution on tissue samples.

Supported by the Masaryk University: project no.   MUNI/A/1421/2021, MUNI/IGA/1208/2021,
MUNI/A/1398/2021, and MUNI/11/ACC/3/2022.