Sublimation application of 5‐chloro‐2‐mercaptobenzothiazole matrix for matrix‐assisted laser desorption/ionization mass spectrometry imaging of mouse kidney

Rationale Sublimation is a solvent‐free technique used to apply a uniform matrix coating over a large sample plate, improving the matrix’s purity and enhancing the analyte signal. Although the 5‐chloro‐2‐mercaptobenzothiazole (CMBT) matrix was introduced years ago, there are no reports of its application via sublimation. We investigated the experimental parameters that are optimal for CMBT matrix sublimation on mouse kidney samples. We also evaluated the stability of the sublimed CMBT matrix under a vacuum environment. Using kidney samples prepared with a sublimated CMBT matrix, we conducted matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) analysis of specific phospholipids (phosphatidylcholine and phosphatidylglycerol in the positive ion mode and phosphatidylinositol in the negative ion mode). We also explored various spatial resolutions (50, 20, and 10 μm) and performed sequential MALDI–hematoxylin and eosin (H&E) staining. Methods The CMBT matrix was applied to kidney samples using a sublimation apparatus connected to a vacuum pump to achieve a pressure of 0.05 Torr. The matrix was then subjected to different temperatures and sublimation times to determine the optimal conditions for matrix application. Subsequently, a Q‐Exactive mass spectrometer equipped with a Spectroglyph MALDI ion source was employed for MALDI‐MSI experiments. Standard protocols were followed for H&E staining after MALDI analysis. Results A matrix thickness of 0.15 mg/cm 2 yielded high‐quality images. The sublimated matrix exhibited minimal loss after approximately 20 h of exposure to a vacuum of 7 Torr, indicating its stability under these conditions. Ion images were successfully obtained at spatial resolutions of 50, 20, and 10 μm. Furthermore, orthogonal histological information was obtained through sequential MALDI‐H&E staining. Conclusions We demonstrate that samples prepared for MALDI‐MSI using sublimation to apply the CMBT matrix yield high‐quality mass spectrometric images of mouse kidney sections. We also provide data for the impact of various experimental parameters on image quality (e.g., temperature, time, matrix thickness, and spatial resolution).