Kamiie Junichi, Aihara Naoyuki, Uchida Yu, Kobayashi Daiki, Yoshida Yutaka, Kuroda Takeshi, Sakaue Motoharu, Sugihara Yutaka, Rezeli Melinda, Marko-Varga György
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan.
Omics Unit, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
MethodsX. 2020 Jan 27;7:100770. doi: 10.1016/j.mex.2019.100770. eCollection 2020.
Typing of amyloidosis by mass spectrometry (MS) based proteomic analysis contribute to the diagnosis of amyloidosis. For MS analysis, laser microdissection (LMD) is used for amyloid specific sampling. This study aimed to establish a method for selectively extracting amyloids from formalin-fixed, paraffin-embedded (FFPE) specimens by organic solvent instead of LMD. The extracts using dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, trifluoroethanol (TFE) or hexafluoro-2-propanol from FFPE brain of alzheimer's disease mouse model generated protein bands on SDS-PAGE, and Aβ was identified in the extract of DMF using mass spectrometry. The extract using DMSO from the kidney of a AA amyloidosis patient produced a protein band in SDS-PAGE. This protein band was identified to be serum amyloid A (SAA) by Western blotting and mass spectrometry. Circular dichroism spectrometry revealed that the secondary structures of Aβ and transthyretin were converted to α-helices from β-sheets in TFE. Our results suggest that organic solvents can extract amyloids from FFPE specimens by converting their secondary structure. This method could eliminate the LMD step and simplified amyloid typing by MS analysis. •DMSO, DMF, methanol, TFE and HFIP can extract Aβ specifically from the FFPE brain of a Alzheimer' disease mouse model.•DMSO can extract SAA specifically from a FFPE section of AA amyloidosis.•Secondary structures of Aβ and transthyretin converted from β-sheet to α-helix in TFE.
基于质谱（MS）的蛋白质组学分析对淀粉样变性进行分型有助于淀粉样变性的诊断。对于MS分析，激光显微切割（LMD）用于淀粉样蛋白特异性采样。本研究旨在建立一种通过有机溶剂而非LMD从福尔马林固定、石蜡包埋（FFPE）标本中选择性提取淀粉样蛋白的方法。使用二甲基亚砜（DMSO）、二甲基甲酰胺（DMF）、甲醇、三氟乙醇（TFE）或六氟异丙醇从阿尔茨海默病小鼠模型的FFPE脑提取物在SDS-PAGE上产生蛋白条带，并且使用质谱在DMF提取物中鉴定出Aβ。使用DMSO从一名AA淀粉样变性患者的肾脏提取物在SDS-PAGE上产生一条蛋白条带。通过蛋白质印迹法和质谱鉴定该蛋白条带为血清淀粉样蛋白A（SAA）。圆二色光谱显示，在TFE中，Aβ和转甲状腺素蛋白的二级结构从β折叠转变为α螺旋。我们的结果表明，有机溶剂可以通过转换其二级结构从FFPE标本中提取淀粉样蛋白。该方法可以省去LMD步骤并简化通过MS分析进行的淀粉样蛋白分型。•DMSO、DMF、甲醇、TFE和HFIP可以从阿尔茨海默病小鼠模型的FFPE脑中特异性提取Aβ。•DMSO可以从AA淀粉样变性的FFPE切片中特异性提取SAA。•在TFE中，Aβ和转甲状腺素蛋白的二级结构从β折叠转变为α螺旋。
