Amyloid-specific extraction using organic solvents

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.

Graphical abstract

Fig. 1

Extraction of amyloid β from 5XFAD mouse brain using organic solvents. (A) SDS-PAGE (silver stain) of extracts from FFPE brains of 5XFAD mouse and C57/B6 mouse with organic solvents. (B) MS/MS spectrum of the doubly charged ion with a specific mass-to-charge ratio 663.34 identified as LVFFAEDVGSNK, which was derived from amyloid βA4.

Fig. 2

(A) Histopathological findings of the kidney of a patient with AA amyloidosis. HE staining (right) shows amorphous material deposition in glomerular and interstitial tissues. Amorphous materials were stained by Congo red (centre) and are immunohistochemically positive against SAA (left) (scale bar: 100 μm). (B) SDS-PAGE (CBB staining) of the extract from FFPE kidney of a patient with AA amyloidosis with DMSO. (C) Western blotting of the extract against SAA. (D) MS/MS spectrum of the doubly charged ion with a specific mass-to-charge ratio 728.37 identified as GPGGAWAAEVISNAR, which was derived from SAA2.