Simple fluorinated moiety insertion on Aβ 16-23 peptide for stain-free TEM imaging.

Silvia Sonzini,Samuel T Jones,Zarah Walsh,Oren A Scherman

doi:10.1039/c4an02278e

Silvia Sonzini, Samuel T Jones + Show 2 more

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https://doi.org/10.1039/c4an02278e

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Abstract

Peptide aggregation and fibre formation are one of the major underlying causes of several neurodegenerative disorders such as Alzheimer's disease. During the past decades the characterisation of these fibres has been widely studied in an attempt to further understand the nature of the related diseases and in an effort to develop treatments. Transmission electron microscopy (TEM) is one of the most commonly used techniques to identify these fibres, but requires the use of a radioactive staining agent. The procedure we report overcomes this drawback through simple addition of a fluorinated moiety to a short Amyloid β sequence via solid phase peptide synthesis (SPPS). This method is synthetically straightforward, widely applicable to different aggregation-prone sequences and, above all, allows for stain-free TEM imaging with improved quality compared to standard imaging procedures. The presence of the fluorinated moiety does not cause major changes in the fibre structure or aggregation, but rather serves to dissipate the microscope's electron beam, thus allowing for high contrast and straightforward imaging by TEM.

Highlights

Several degenerative diseases, such as Alzheimer’s,1 Parkinson’s,2 Huntington’s disease3 and type II diabetes4 are related to an accumulation of amyloid fibrils.5 The proteins composing these fibrils vary in each of the different diseases, but there are substantial similarities in their structural properties
The standard procedure for transmission electron microscopy (TEM) imaging, consists of staining the fibres with a radioactive uranyl acetate solution,8 which allows the fibres to be imaged as the uranyl acetate helps to dissipate the electron beam
Building on previous work from our group21 we report, the incorporation of an N-terminal fluorinated moiety (F) via solid phase peptide synthesis (SPPS) that allows stain-free TEM to be performed on peptide fibres

Summary

Introduction

Several degenerative diseases, such as Alzheimer’s,1 Parkinson’s,2 Huntington’s disease and type II diabetes are related to an accumulation of amyloid fibrils. The proteins composing these fibrils vary in each of the different diseases, but there are substantial similarities in their structural properties. The structure of Amyloid β (Aβ) fibrils has been studied with numerous techniques to capture as much information as possible on their aggregation pathway and further understand the development of the related disease.8–15 These fibrils have been investigated for their role in Alzheimer’s disease, but they have been gathering increasing interest on account of their mechanical and adhesive properties and their natural propensity to self assemble.. The standard procedure for TEM imaging, consists of staining the fibres with a radioactive uranyl acetate solution, which allows the fibres to be imaged as the uranyl acetate helps to dissipate the electron beam This method, even if widely applied, can be misleading since the microscope images show only regions that interact with the staining solution and not necessarily the peptide itself. In our case only the material ( peptide) containing the fluorine moiety will be seen in the TEM, since no additional staining technique is applied (Fig. 1)

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