Fülöp Annabelle, Marsching Christian, Barka Frederik, Ucal Yasemin, Pfänder Pauline, Opitz Christiane A, Barka Günes, Hopf Carsten
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany.
SunChrom Wissenschaftliche Geräte GmbH, 61381 Friedrichsdorf, Germany.
Pharmaceuticals (Basel). 2022 Nov 3;15(11):1356. doi: 10.3390/ph15111356.
On-tissue enzymatic digestion is a prerequisite for MALDI mass spectrometry imaging (MSI) and spatialomic analysis of tissue proteins and their -glycan conjugates. Despite the more widely accepted importance of -glycans as diagnostic and prognostic biomarkers of many diseases and their potential as pharmacodynamic markers, the crucial sample preparation step, namely on-tissue digestion with enzymes like PNGaseF, is currently mainly carried out by specialized laboratories using home-built incubation arrangements, e.g., petri dishes placed in an incubator. Standardized spatially confined enzyme digests, however, require precise control and possible regulation of humidity and temperature, as high humidity increases the risk of analyte dislocation and low humidity compromises enzyme function. Here, a digestion device that controls humidity by cyclic ventilation and heating of the slide holder and the chamber lid was designed to enable controlled micro-condensation on the slide and to stabilize and monitor the digestion process. The device presented here may help with standardization in MSI. Using sagittal mouse brain sections and xenografted human U87 glioblastoma cells in CD1 nu/nu mouse brain, a device-controlled workflow for MALDI MSI of -glycans was developed.
组织上的酶解消化是基质辅助激光解吸电离质谱成像（MSI）以及对组织蛋白及其聚糖共轭物进行空间组学分析的前提条件。尽管聚糖作为许多疾病的诊断和预后生物标志物的重要性得到了更广泛认可，并且具有作为药效学标志物的潜力，但关键的样品制备步骤，即使用诸如肽：N-聚糖酶F等酶进行组织上的消化，目前主要由专业实验室使用自制的孵育装置进行，例如放在培养箱中的培养皿。然而，标准化的空间受限酶解消化需要精确控制并可能调节湿度和温度，因为高湿度会增加分析物错位的风险，而低湿度会损害酶的功能。在此，设计了一种通过对载玻片架和腔室盖进行循环通风和加热来控制湿度的消化装置，以实现载玻片上的可控微冷凝，并稳定和监测消化过程。这里介绍的装置可能有助于MSI的标准化。利用矢状面小鼠脑切片以及在CD1裸鼠脑内移植的人U87胶质母细胞瘤细胞，开发了一种用于聚糖的MALDI MSI的装置控制工作流程。
