Gustavo Gonçalves Silva¹, Francisca Kaline Ferreira Pereira¹, Amanda Braga de Figueiredo¹, Carlos Eduardo dos Santos Ferreira², Lívia Schiavinato Eberlin³, Kenneth John Gollob¹

The MasSpec Pen is an innovative device designed for rapid molecular analysis of biological tissues via mass spectrometry. Developed as a tool for real-time tissue characterization, it employs a gentle aqueous extraction method using a pen-shaped probe. This process allows for the collection of surface molecules without causing structural damage or disrupting cellular organization. The extracted compounds are then promptly analyzed in the mass spectrometer, eliminating the need for conventional separation steps. This enables the detection of a wide range of biomolecules, including metabolites, lipids, and proteins, facilitating tissue-specific and spatially informative molecular profiling. The objective of this study is to apply the MasSpec Pen to mouse brain tissue analysis - a complex biological matrix - to collect mass spectral data capable of differentiating distinct anatomical brain regions based on their unique molecular signatures. Brain samples were obtained from female C57BL/6 mice (CEUA: 5635/2023), sectioned sagittally, and stored at -80°C. Prior to analysis, sections were thawed for five minutes and placed on glass slides. The MasSpec Pen was interfaced with an Orbitrap Exploris 120 mass spectrometer operating in full scan mode at 120,000 resolution, covering a scan range of 150–1000 m/z in both positive and negative ionization modes. A custom ACG target was set, and two microscans were acquired per scan. A 3 mm pen tip was used with a five-second sampling time followed by a ten-second aspiration step. Each brain region was analyzed in triplicate. Our results revealed a complex repertoire of biomolecules extracted from the brain tissue, displaying a diverse range of ions across the mass spectrum. Spectral data from gray and white matter regions showed distinct molecular profiles, with several ions differentially represented between these areas. Furthermore, tentative identification of molecular ions based on high mass accuracy measurement suggested the presence of various lipid classes, including fatty acids, ceramides, glycerophospholipids, and triacylglycerols, as well as amino acid derivatives. This approach enabled the direct detection of a wide range of biologically relevant molecules. The high-resolution mass spectrometric data obtained allowed for sensitive and specific molecular profiling of distinct brain regions, revealing consistent spatial patterns in metabolite and lipid distribution. These results support the use of the MasSpec Pen as a tool for tissue molecular characterization, offering a rapid assessment of region-specific biochemical composition. Furthermore, these findings support its potential application during intraoperative analysis of tumor tissues, serving as an innovative tool to differentiate healthy from cancerous tissues at the molecular level - a key focus of our next steps.

Agradecimentos: FAPESP; Instituto Israelita de Ensino e Pesquisa Albert Einstein; Hospital Israelita Albert Einstein.