Click and detect: a new generation of tagged probes for customed targeted multiplex MS imaging

By bringing a spatial dimension, MS Imaging (MSI) has shown to be a game changer for biology and clinics. However, despite steady progress in sensitivity many analytes remain invisible to MSI. This issue can be circumvented by the development of a targeted approach where a modified probe enabling MS detection is used in combination with conventional affinity-based imaging methods. Hence, the TagMass concept was previously demonstrated with oligonucleotide probes for imaging mRNA through in situ hybridization (ISH) and antibodies for imaging proteins via immunohistochemistry (IHC). Henceforth we have developed a new generation of tags that can be easily used to customize various probes and perform multiplex targeted MSI thanks to biorthogonal conjugation. This novel generation of tags is not only easily coupled to any probe thanks to the click-chemistry but is also designed to enable both photo- or chemically cleavage which can then be used in MALDI- or DESI-MSI. Hence the produced probes are composed of a linker, a photo- or chemically cleavable group, and a tag chosen to show good MS detection. Based on this principle, more than 10 novel tags were synthesized with different, but close in mass, chemical functions to get a similar analytical behavior and an accurate quantification level These tags were characterized by MS and NMR, and their cleavage yield was assessed alone and mixed. Then different tagged probes were produced from primary antibodies using bioconjugation followed by strain-promoted azide-alkyne click chemistry (SPAAC) reaction to perform targeted multiplex in MALDI- and DESI-MSI. First, the different tags were compared on the same antibody (anti-GFAP). Then different primary antibodies directed against various targets present in the rat brain (including for example GFAP as an astrocytic marker, NeuN for neurons, Myelin for oligodendrocytes, and tubulin beta-3 as a marker of stem cells and neural progenitors) were modified to perform multiplexing and cross-validated by Fluorescence IHC (FIHC). Some of the antibody probes were also functionalized with a chemically cleavable tag and tested in MALDI and DESI. Additionally, some tags were used to functionalize aptamers considering the various advantages of aptamers over antibodies in terms of specificity, binding affinity, and cost. Finally, a proof-of-concept maw made by customizing other probes directed against markers for ovarian and breast cancers such as PDL-1, CD47, TROP-2, Reg-Alpha, HER2, and markers of the immune cells.