Yan Yan,Jiaying Liu,Yingjian Deng,Yu Tao,Xinxin Li,Chenhui Du,Kun Yang,Ruiping Zhang
Objectives:Insomnia is a highly prevalent sleep disorder involving complex neurochem-ical dysregulation; however, the spatial distribution of neurotransmitters and small-molecule metabolites across distinct brain microregions in the insomniac state re-mains poorly characterized. This study aimed to map region-specific metabolic perturba-tions in situ in a p-chlorophenylalanine (PCPA)-induced insomnia rat model using opti-mized matrix-assisted laser desorption/ionization mass spectrometry imaging (MAL-DI-MSI) integrated with targeted metabolomics validation.Methods:On-tissue chemical derivatization MALDI-MSI was optimized using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix on a Bruker tims TOF flex mass spectrometer. The rats received PCPA (400 mg/kg, intraperitoneal) for three days to establish the insomnia model. Metabolite identifi-cation was conducted using MetaboScape® software (2020b) and the Human Metabolome Database. Ultra-performance liquid chromatography–tandem mass spectrometry was em-ployed to quantify nine key neurotransmitters and metabolites across six brain microre-gions. Key synthetic enzyme expression was evaluated by immunofluorescence and West-ern blotting analyses.Results:TMP-TFB-derived brain slices clearly showed the distribu-tion of neurotransmitters in brain microregions. MALDI-MSI demonstrated that the spatial distribution and abundance of eight neurotransmitters were disturbed in brains of PCPA-induced insomniac rats. A total of 346 metabolites were characterized across six brain microregions (cerebellum, cortex, hippocampus, brainstem, hypothalamus, and stri-atum), with principal coordinate analysis revealing clear metabolic separation between control and insomniac rats. PCPA treatment markedly disrupted tryptophan and tyrosine metabolism, evidenced by decreased 5-HT, 5-HTP, and 5-HIAA, alongside region-specific alterations in DA, NE, HVA, and Ach. Additionally, GABA levels decreased in the hippo-campus, striatum, and hypothalamus, whereas glutamate increased throughout the brain. Targeted metabolomics validated the MSI findings, and Bland–Altman analysis confirmed good consistency between the two analytical platforms. PCPA further disturbed the meta-bolic enzymes MAOA, DDC, and TPH2 within the Trp-5-HTP-5-HT-5-HIAA metabolic pathway in the brainstem and TYH and DBA within the tyrosine-DA-NE metabolic pathway in the striatum.Conclusions:This study demonstrates that region-specific altera-tions in tryptophan and tyrosine metabolism pathways provide mechanistic insights into insomnia pathogenesis and potential therapeutic targets.
Tianyu Ma,Chenglong Sun,Yuhao Han,Lanping Guo,Luqi Huang,Xiao Wang
Gastrodiae Rhizoma is a commonly used plant material for both medicine and food in China for thousands of years. Steaming as the main pre-processing method of Gastrodiae Rhizoma is the key to its quality formation. In this study, we established a high-coverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method to visualize the spatial distribution of phenols in Gastrodiae Rhizoma . By optimizing 1,5-diaminonaphthalene as MALDI matrix in negative mode, the “spatial-temporal-content” changes of 13 phenols including 11 parishins in Gastrodiae Rhizoma during the steaming process were imaged. We also characterized the activity of two hydrolases related to parishins degradation during the steaming. Moreover, the degradation pathways of parishins in Gastrodiae Rhizoma were summarized. The integration of spatially-resolved parishins and corresponding hydrolases information revealed the scientific connotation of steaming as the processing method of Gastrodiae Rhizoma is to inactivate hydrolases and protect parishins, which provided theoretical support for the quality improvement and the standardized processing of Gastrodiae Rhizoma .
Matrix deposition plays a critical role in image quality of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). To improve the ionization efficiency and overcome the limitation of traditional matrix deposition methods in the face of difficult-to-sublimate or difficult-to-dissolve matrix, covalent organic frameworks (COFs) named COF-DhaTab was successfully synthesized and firstly used as matrix film. It was fabricated by imprinting of sieved COF-DhaTab powder on the surface of a double-sided adhesive tape. Outstanding reproducibility and uniformity of COF-DhaTab film were demonstrated by relative standard deviation (RSD) within 8.37% and 7.71% from dot-to-dot and plate-to-plate, respectively. With the introduction of double-sided adhesive tape, water contact angle (WCA) of COF-DhaTab film increased from 55° to 141°, resulting in significant suppression of analyte diffusion. Moreover, the intensity of potassium perfluorooctanic sulfonate (PFOS, C 8 F 17 SO 3 − , m/z 498.93) was 9.3 × 10 5 , more than six hundred times higher than that using DHB matrix. This enhancement was attributed to the rough surface and multiple branches of the synthesized COF-DhaTab. To verify the ability of COF-DhaTab film as substrate, the spatial distribution of PFOS in zebrafish, rat liver and kidney tissues was explored. Superior imaging capability was displayed with high-spatial resolution and reliable location distribution. These results not only demonstrate the outstanding ability of COF-DhaTab as matrix for MALDI-MS and MALDI-MSI, but also provide a facile approach for fabrication of novel matrix films for MALDI-MSI.
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