Kasarla Siva Swapna, Fecke Antonia, Smith Karl William, Flocke Vera, Flögel Ulrich, Phapale Prasad
Leibniz-Institut für Analytische Wissenschaften─ISAS─e.V., Otto-Hahn-Str. 6b, Dortmund 44227, Germany.
Experimental Cardiovascular Imaging, Institute for Molecular Cardiology, Heinrich Heine University Düsseldorf, Düsseldorf 40225, Germany.
Anal Chem. 2025 May 27;97(20):10720-10728. doi: 10.1021/acs.analchem.5c00620. Epub 2025 May 13.
Imaging small polar metabolites and analyzing their dynamics with stable isotope-labeled (SIL) tracing through various biochemical pathways, including the citric acid (TCA) cycle, glycolysis, and amino acid metabolism, have gained substantial interest over the years. However, imaging these small polar metabolites across different tissue types is limited due to their lower ionization efficiencies and ion suppression from larger abundant biomolecules. These challenges can be further exacerbated with SIL studies, which require improvements in sample preparation and method sensitivity. Solvent pretreatments before matrix application on a tissue section have the potential to improve the sensitivity of metabolite imaging; however, they are not yet widely optimized across tissue types. Furthermore, there is a recurring concern about metabolite delocalization from such wash treatments that require "spatial validation". Here, we optimized a simple "basic hexane" wash method that improved sensitivity up to several folds for a broad range of polar and H-labeled metabolites across five different mouse organ tissues (kidney, heart, brain, liver, and brown adipose tissue). Notably, we provided region-specific quantification of 51 metabolites using laser microdissection (LMD)-LC-MS/MS to validate their localization observed in MALDI-MSI analysis after the basic hexane wash. Overall, we reported an improved MALDI-MSI sample pretreatment method with a "spatial validation" workflow for sensitive and robust imaging of polar metabolite distributions in mouse organs.
多年来，利用稳定同位素标记（SIL）示踪技术对小极性代谢物进行成像，并分析它们在包括柠檬酸（TCA）循环、糖酵解和氨基酸代谢等各种生化途径中的动态变化，已引起了广泛关注。然而，由于这些小极性代谢物的电离效率较低，且会受到大量丰富生物分子的离子抑制，因此跨不同组织类型对其进行成像受到限制。在SIL研究中，这些挑战可能会进一步加剧，因为这需要改进样品制备和方法灵敏度。在组织切片上施加基质之前进行溶剂预处理有可能提高代谢物成像的灵敏度；然而，尚未针对不同组织类型对其进行广泛优化。此外，对于这种需要“空间验证”的洗涤处理导致代谢物离位的问题一直存在担忧。在这里，我们优化了一种简单的“碱性己烷”洗涤方法，该方法可将五种不同小鼠器官组织（肾脏、心脏、脑、肝脏和棕色脂肪组织）中广泛的极性和H标记代谢物的灵敏度提高数倍。值得注意的是，我们使用激光显微切割（LMD）-LC-MS/MS对51种代谢物进行了区域特异性定量，以验证碱性己烷洗涤后在MALDI-MSI分析中观察到的它们的定位。总体而言，我们报告了一种改进的MALDI-MSI样品预处理方法，该方法具有“空间验证”工作流程，可以对小鼠器官中的极性代谢物分布进行灵敏且可靠的成像。
