Session: Session 4

Click and Detect: A New Generation of Tagged Probes for Customable Targeted Multiplex MS Imaging

MS Imaging (MSI) has revolutionized biology and clinics by adding a spatial dimension to mass spectrometry (MS). However, many analytes remain undetectable due to limited abundance, poor ionization yield, or high molecular weight. This issue can be addressed by using probes designed to recognize targets and be detectable by MS. We developed customizable probes for multiplex targeted MSI through bioorthogonal conjugation.



Probes are modified with a reporter group, including a linker, a photo-cleavable group, and a tag for good MS detection. The tagged probe recognizes a target in tissue, and UV light induces cleavage. The released tag is detected and imaged by MSI, revealing the target's distribution. Various reporters with similar mass chemical functions were synthesized and grafted onto the probe after bioorthogonal conjugation. These tagged probes are used for multiplex targeted MALDI.



Synthesized reporters were characterized by NMR and MS for purity and structure confirmation. They were then tested under UV to determine photocleavable yields, photocleavage time, and their MS response, both alone and mixed. Over 10 different probes were synthesized starting with antibodies using bioconjugation, followed by strain-promoted azide-alkyne click chemistry (SPAAC) to graft several reporters onto the probe and amplify the signal. Each reporter was grafted onto an anti-rat primary antibody for immunohistochemistry, targeting GFAP in rat brain tissue sections. MALDI MS images showed similar distribution and detection efficiency among different tags. Reporters were then used to functionalize primary antibodies against rat brain targets, including GFAP (astrocytes), NeuN (neurons), Myelin (oligodendrocytes), and tubulin beta-3 (stem cells and progenitors), resulting in detectable signals in a multiplex experiment. 



Finally, a proof-of-concept was made by customizing probes for breast cancer markers such as PDL-1, CD47, FOX-P3, Reg-Alpha, HER2, and markers of immune cells.