Amyloid-specific extraction using organic solvents

Junichi Kamiie; Naoyuki Aihara; Yu Uchida; Daiki Kobayashi; Yutaka Yoshida; Takeshi Kuroda; Motoharu Sakaue; Yutaka Sugihara; Melinda Rezeli; György Marko-Varga

doi:10.1016/j.mex.2019.100770

Amyloid-specific extraction using organic solvents

MethodsX. 2020 Jan 27:7:100770. doi: 10.1016/j.mex.2019.100770. eCollection 2020.

Authors

Affiliations

¹ Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan.
² Omics Unit, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
³ Department of Structural Pathology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
⁴ Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
⁵ Laboratory of Veterinary Anatomy II, School of Veterinary Medicine, Azabu University, Sagamihara, Japan.
⁶ Division of Clinical Protein Science & Imaging, Department of Biomedical Engineering, Lund University, Lund, Sweden.

Abstract

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.

Keywords: Amyloid; Amyloid-specific extraction from FFPE specimens using organic solvents; Formalin-fixed; Organic solvents; Paraffin-embedded; Protein extraction.