PROGRAMME

Upon its development (Karas et al., 1985), Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry (MS) has long been leveraged by researchers for the detection of larger biologically relevant molecules, such as, proteins, glycans and peptides (van Kampen et al., 2011, Shariatgorji et al., 2012). Analysis of small molecules such as metabolites with MALDI, however, particularly in the case of imaging, is lately of increasing importance for various applications, such as biological and clinical research. This is mostly owning to the fact that imaging of a tissue sample can provide insightful information on the spatial distribution and abundance of biological relevant molecules, involved in the biology of diseases, and lead to the discovery of new biomarkers (Vaysse et al., 2017, Chungtai and Heeren, 2010, Giampà et al., 2016). One of the most crucial parameters, affecting the MS signal, and hence the MALDI analysis, is the matrix type choice (Giampà et al., 2016, Horatz et al, 2021). The matrix usually crystallizes with the sample’s analytes, absorbs the laser energy, thus causing the desorption and then ionization of the analytes (Shariatgorji et al., 2012). Classic matrices commonly employed in MALDI techniques are generally small organic molecules (SOMs) that exhibit prerequisite characteristics for MALDI analysis, e.g., strong laser irradiation absorption (Horatz et al., 2021). Some examples are α-Cyano-4-hydroxycinnamic acid (α-CHCA), 2,5-Dihydroxybenzoic acid (2,5 – DHB), 9-Aminoacridine (9AA), Sinapinic acid (SA) etc. Classic MALDI matrices have been previously successfully utilized in low molecular weight compound (LMWC) imaging techniques of various classes, such as lipids (Fu et al., 2019), metabolites (Bhandari et al., 2015) and others. One of the biggest drawbacks of employing SOMs as matrices, however, is the hindrance and complication of analyte detection in the lower mass ranges. This is mostly attributed to the fact that the small organic compounds can desorb and be ionized, thus providing large interfering matrix-related background signals (fragments, clusters etc.) at the m/z mass range of the analytes of interest (Horatz et al, 2021, Horatz et al., 2018, Smirnov et al., 2004), thus interfering with their detection. For this reason, MALDI analysis of small molecules has, for a long time, been considered problematic or even non-feasible. One of the strategies developed to overcome the aforementioned problem, is the exploitation of matrices that exhibit a higher molecular weight than the range of interest. Such compounds for example are porphyrins and conjugated polymers (Qiao and Lissel, 2021). Hitherto, a handful of polymeric matrices have been applied to LMWC MALDI analysis, providing promising results ( Horatz et al, 2018, Horatz et al., 2019). More specifically K. Horatz et al., successfully applied poly{[N,N’-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis- (dicarboximide)-2,6-diyl]-alt-5,5’(2,2’-bithiophene)} (PNDI(T2)), poly(3-dodecylthiophene-2,5-diyl) (P3DDT), poly{[2,3-bis(3-octyloxyphenyl)quinoxaline-5,8-diyl]-alt- (thiophene-2,5-diyl)} (PTQ1), and poly{[N,N’-bis(2-octyldodecyl)-isoindigo-5,5’-diyl]-alt-5,5’(2,2’-bithiophe -ne)} (PII(T2)), for the detection of standard LMWC analytes. All matrices were found to be MALDI silent, as well as rare dual-mode matrices, providing results comparable to those obtained by employing classic MALDI matrices. Additionally, P3DDT was also successfully used as an imaging matrix for rat tissue morphology visualization (Horatz et al., 2018). Accordingly, K. Horatz et al. efficiently employed newly synthesized amorphous polymers for the detection of LMWC such as reserpine and coumaphos. The acquired results of the study demonstrated the good performance of these copolymers as matrices and their potential as dual-mode, MALDI silent matrices. The measured intensities of LMWCs with these co-polymers as matrices also proved to be equal or higher when compared to those of the semicrystalline polymers tested (Horatz et al., 2019). The above-mentioned results highlight the appealing and promising potential of polymer exploitation as matrices in MALDI-MS workflows. However, the application of polymer-based matrices remains not fully explored yet. Hence, the aim of this study was to: 1) further investigate the potential of various polymers for efficient LMWC (e.g. pharmaceuticals) ionization and detection, 2) directly compare the acquired results with those obtained from classic matrices applications (e.g., 2,5-DHB, α-CHCA) and finally, 3) develop high resolution mass spectrometric (MALDI-HRMS) analytical protocols and workflows for small molecule analysis. For this purpose, newly synthesized and characterized polymer-based MALDI matrices were developed. These matrices were designed to effectively absorb the laser wavelength of interest and demonstrate other desired physicochemical properties. The ionization potential of these matrices on various LMW analytes was tested on a ground steel MPS 384 plate, on a timstof flex (Bruker, Germany) instrument. Different sample preparation protocols and instrumental parameters were tested in order to choose the conditions that yield the optimum results. The results showcase the potential of polymeric matrix applications for future detection of LMWCs, as well as bottlenecks that might need to be taken into consideration in MALDI-HRMS workflows.