Ito Masatoshi, Fujii Natsuko, Koguchi Shinichi, Nagata Eiichiro
Department of Neurology, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Department of Legal Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan.
Methods Mol Biol. 2025;2972:81-93. doi: 10.1007/978-1-0716-4799-8_7.
The direct detection of InsP (diphosphoinositol pentakisphosphate) and InsP (bis-diphosphoinositol tetrakisphosphate), known as inositol pyrophosphates (PP-InsPs), in mammalian specimens faces technical difficulties owing to their characteristic chemical properties and minute quantities in mammalian tissues. We developed an analytical protocol to sensitively and directly detect PP-InsPs and their precursor, inositol hexakisphosphate (InsP), using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). This analytical protocol, coupled with high-purity synthetic standards, allows for the absolute quantitation of these analytes in cellular samples as well as in various organs and tissues obtained by autopsies of animals and humans, offering an effective option to study PP-InsP functions in mammalian physiology and pathology. Herein, we describe this protocol in detail, from the sample preparation procedure throughout the column regeneration after analysis, along with several notable cautionary points.
由于二磷酸肌醇五磷酸（InsP）和双二磷酸肌醇四磷酸（InsP）（即肌醇焦磷酸，PP-InsPs）具有独特的化学性质且在哺乳动物组织中的含量极少，因此在哺乳动物样本中直接检测它们面临技术难题。我们开发了一种分析方法，利用亲水作用液相色谱-串联质谱法（HILIC-MS/MS）灵敏且直接地检测PP-InsPs及其前体肌醇六磷酸（InsP）。该分析方法结合高纯度合成标准品，能够对细胞样本以及通过动物和人类尸检获得的各种器官和组织中的这些分析物进行绝对定量，为研究PP-InsP在哺乳动物生理和病理过程中的功能提供了一种有效的选择。在此，我们详细描述该方法，从样品制备过程到分析后的柱再生，以及几个值得注意的注意事项。
