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标题：On-slide tissue digestion for mass spectrometry based glycomic and proteomic profiling
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作者：Rekha Raghunathan ; Manveen K. Sethi ; Joseph Zaia 等
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2019
卷号：6
页码：2329-2347
DOI：10.1016/j.mex.2019.09.029
语种：English
出版社：Elsevier
摘要：Graphical abstractDisplay OmittedAbstractWe describe a protocol for glycomic and proteomic profiling that uses serial enzyme digestions from the surface of fresh frozen or fixed tissue slides. The abundances of the extracted glycans and peptides are determined using liquid chromatography-mass spectrometry. In a typical experiment, our method quantifies 14 heparan sulfate disaccharides, 11 chondroitin sulfate disaccharides, 50N-glycan compositions and approximately 1200 proteins from a 1.8 mm circle, on the surface of a fresh frozen tissue slide from rat brain. Each enzymatic digestion is incubated overnight with direct application of enzyme on the tissue surface. Overall, the sample preparation process for multiple tissue slides takes a day per biomolecule class. This protocol saves time by simultaneous digestion of largeN-glycans and small HS disaccharides and subsequent separation using size exclusion chromatography. Compared to wet tissue analysis, this method requires less time by a factor of two. By comparison, MALDI-imaging provides higher spatial resolution of glycans and proteins but lower depth of coverage. MALDI dissociates fragile glycan substituents including sulfates and is not recommended for analysis of glycosaminoglycans (GAGs).
关键词：Glycomics;Proteomics;On-slide;Tissue digestion;Mass spectrometry;FFPE profiling;Fresh frozen profiling;LC–MS/ MS;Nano-HILIC;Reversed phase