Epitope mapping

Epitope mapping by PepChipOmics*Søren Buus1 Gustav A. Röder1, Sune Justesen1, and Claus Schäfer-Nielsen2

1:Department of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen2: Schafer-N, Copenhagen*The PepChipOmics project is funded under the 7th EU framework programme (FP7)

Abstract:Peptides have emerged as indispensable tools in the elucidation of protein function and interaction. Chemical synthesis of peptides has now become a

standard procedure and synthetic peptides can currently be produced for prices down to about 30 EURO for a 10 amino acid long peptide. Even with such

low prices, the costs of peptides for a comprehensive screening program can easily amount to hundreds of thousands of EURO, and the cost of the

screening program may itself be prohibitive due to the cumbersome maneuvers that are often involved in assaying many individual peptides. Thus, the

pharmaceutical and biotechnology industries, as well as academia, encounter significant economic and logistical barriers when they attempt to apply

peptide-based screening programmes in the exploitation of “-omics” information.

In this project we will use digital mirror devices (DMD) in combination with a photosensitive peptide synthesis to generate stamp sized peptide arrays.

Currently arrays of more than individual 12.000 peptides can be constructed but the project aims at more than 100.000.

In the current example we will show how the peptide chip could be used to map the epitope of a monoclonal murine antibody specific for the immunological

important protein Tapasin

A: Digital Mirror Device (DMD), micron sized mirrors can tilt app. 10º. B: Illumination

of pixels on chip by DMD.

C: Step 1. All exposed aminogroups in the growing peptide chain is protected by a

photosensitive group. Step 2- 10. Upon UV illumination deprotected chains can be

elongated with the desired amino acid. Step 11+12 At the end of a cycle all protective

Fmoc groups are exchanged with a photosensitive protection group, to start a new

elongation cycle.

2 µm

A B

C

Construction of peptide chip

The amino acid sequence of

Tapasin (428 amino acids) was

divided into 12-mer peptides

overlapping by 11 amino acids.

Each of these peptides were

further alanine substituted at

each position to create a full

alanine scan of each individual

peptide.

Peptide chip layout

The produced Tapasin chip was incubated with a monoclonal antibody produced from a mouse

immunized with a truncated tapasin (amino acids 1-87). After detection with a secondary Cy3

marked anti-murine antibody (A), the chip was assayed in a fluorescence microscope (B). The

sliding truncations revealed a single strong signal peak centered on the sequence LDPEL (C),

corresponding to human tapasin residues 40-44, that is placed in the MHC I contact interface

(E+F yellow). Analysis of the alanine scan also revealed the LDPEL core epitope and some

contribution from 6 amino acids flanking the core epitope (F). A peptide LDPELYLSVHD was

synthesized and, in a biochemical assay, shown to inhibit the interaction between the antibody

and Tapasin fragment with an inhibitory constant of 3 nM (H), indicating a strong interaction

between monoclonal antibody and tapasin.

B C

D E

F G

A