9 aminoacridine hydrochloride monohydrate 9 aa

MALDI matrices, including 2.5-dihydroxybenzoic acid (DHB) and 9-aminoacridine hydrochloride monohydrate (9-AA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium trifluoroacetate (NaTFA, Sigma–Aldrich) at 0.1 mg mL⁻¹ (ACN/H₂O 50:50 [v/v]) was used for external calibration before each analysis.

The MALDI-FT-ICR-MSI analyses were performed as previously described^{40 (link)–42 (link)}. In brief, FFPE heart samples were cut into 5 μm sections on a microtome (Leica) and mounted onto indium-tin-oxide coated conductive glass slides (Bruker Daltonik, Bremen, Germany). The FFPE sections were incubated at 60 °C for one hour and deparaffinized in xylene (twice for 8 min). The matrix solution consisted of 10 mg/ml 9-aminoacridine hydrochloride monohydrate (9-AA) (Sigma-Aldrich, Germany) in water/methanol 30:70 (v/v). SunCollect sprayer (Sunchrom, Friedrichsdorf, Germany) was used for matrix application. The flow rates were 10, 20, 30 and 40 μl/min, respectively, for the first four layers. The other 4 layers were performed at 40 μl/min. The MALDI-MSI measurement was performed on a 7 T Solarix XR FT-ICR mass spectrometer (Bruker Daltonik, Bremen, Germany) in negative ion mode using 50 laser shots per spot at a frequency of 1000 Hz. The MALDI-MSI data were acquired over a mass range of m/z 75−1000 Da with 50 μm lateral resolution. MALDI mass spectra were root mean square normalized using SCiLS Lab (Bruker Daltonik, Bremen, Germany), and exported as imzML files. MS peak annotation was performed with Human Metabolome Database^{43 (link)} (http://www.hmdb.ca/) and METASPACE^{44 (link)} (https://metaspace2020.eu/).