P4-076: Immobilization of β-amyloid peptide on a chip and characterization of its interaction with proteins via atomic force microscopy and mass spectrometry

Abstract

Here we describe a strategy using self assembled monolayer (SAM), atomic force microscopy (AFM) and mass spectrometry (MS) to identify attachment of peptide on gold chip and specific peptide–protein interaction. The β–amyloid is a self–aggregating protein found in Alzheimer's disease and is thought to play a major role in the disease process. The methodology of self–assembled monolayers was employed to functionalize gold surfaces with alkanethiol. In this study, β–amyloid (1–40) was synthesized with N–terminal cystein residue in order to achieve a specific attachment to the alkanethiol modified gold surface. Attachment of β–amyloid was characterized using AFM methodology. A modification of the surface corresponding to the peptide attachment was clearly observed. Size measurement of the molecule corresponded to the theoretical expected size of the β–amyloid peptide. Besides, the exact mass of the β–amyloid peptide was observed by MS experiment directly on the gold surface. This peptide array was used to characterize specific interactions between β–amyloid–antibody. Images from the interaction of β–amyloid –antibody were taken by AFM. Profile of the images and size measurement of the complex showed clear differences. Chemiluminescence was used to confirm the presence of the antibody and so the formation of the complex. We are studying now the ability of this β–amyloid–chip to interact specifically with proteins in complex protein extract. Moreover, we would try to identify specific interaction by MS as such an affinity capture surface should avoid protein loss and considerably simplify sample preparation for mass spectrometry analysis. High sensitivity of mass spectrometry should be well suited to this on–chip study.