Aminated cinnamic acid analogs as dual polarity matrices for high spatial resolution MALDI imaging mass spectrometry

We have developed a new class of dual polarity molecules for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) capable of sub 10 µm spatial resolution on commercial instruments. Aminated cinnamic acid analogs (ACAA) were tested for vacuum stability, absorption at 355 nm, crystal size, and molecular coverage. Among them, 4-aminocinnamic acid (ACA) and 4-(dimethylamino)cinnamic acid (DMACA) were found to perform at similar or superior levels for lipid IMS compared to conventional MALDI matrices. ACA generated few in-source fragments, leading to better visualization of thermally labile molecules such as gangliosides. ACA was compared to 2,5-dihydroxyacetophone (DHA) for ganglioside imaging using murine brain tissue. ACA outperformed DHA by generating higher-quality MALDI images due to its higher sensitivity for ganglioside and higher extinction coefficient at 355 nm. ACA also produces a more accurate ganglioside molecular signature with reduced in-source fragmentation, minimizing the production of GM1-like fragments from other gangliosides compared to DHA. Due to its strong extinction coefficient at 355 nm and its vacuum stability, DMACA was a prime candidate for dual polarity phospholipid MALDI IMS at high spatial resolution. DMACA outperformed both 1,5-diaminonaphtalene (DAN) and DHA at their respective laser power in terms of intensity, signal-to-noise, and annotations. DMACA also allows for lower laser power to be used without compromising sensitivity, which allowed a reduction in the spot size from ~6 µm to ~4.5µm. This lower laser burn pattern allows true 5 µm spatial resolution MALDI IMS without oversampling while maintaining high quality signal from a commercial timsTOF fleX instrument without any modifications. Optimizations and final MALDI IMS were performed on murine tissue sample and human kidney sample from the Human BioMolecular Atlas Program (HuBMAP).