XL-MS Direct Comparison with X-Ray Crystallography Epitope Binding Sites Case Study



Lilit Simonyan1, Mamadou Sy1, Ryan Wenzel2, David Lutje-Hulsik3, Hans van Eenennaam3, Alexis Nazabal4


  1. CovalX Analytics SA, 146 rue Léo Saignat, 33076, Bordeaux, France
  2. CovalX Instruments Inc, 999 Broadway, Saugus, MA 01906, USA
  3. Aduro Biotech Europe, Kloosterstraat 9, 5349 AB, Oss, The Netherlands
  4. CovalX AG, Ausstellungsstrasse 36, CH-8005, Zürich, Switzerland


Epitope mapping is one of the key structural analysis during the development of new therapeutic monoclonal antibodies. The characterization of the molecular interface between an antibody and its target antigen is critical, not only for a patent purpose but also for understanding the mechanism of action of the drug itself. If linear epitopes are relatively easy to resolve, conformational epitopes are still challenging to characterize, mainly due to the resolution required for such analysis. We are presenting the use of chemical cross-linking and high resolution mass spectrometry for conformational epitope mapping of Ipilimumab, Nivolumab, and Pembrolizumab monoclonal antibodies.

CovalX Technology Used (Click each option to learn more)

Epitope Mapping



XL Kits


The Method

  1. Each samples were tested for aggregation and noncovalent multimers by chemical cross-linking and High-Mass MALDI ToF MS.
  2. Cross-linking conditions were optimized for each complex: Ipilimumab/CTL4; Nivolumab /PD-1 and Pembrolizumab/PD-1.
  3. Each cross-linked complexes were subjected to proteolysis using five different enzymes.
  4. Cross-linked peptides generated were analysis using nLC/orbitrap MS/MS analysis. The data generated were analyzed by Xquest and Stavrox softwares in order to detect the cross-linked between peptides of the antibody and peptides of the antigen.

The Results

To see the graphic analysis,

Each sample, antibody/antigen mixtures were prepared and analyzed by High-Mass MALDI ToF mass spectrometry (blue traces) (HM4, CovalX). Then each sample was cross-linked (K200, CovalX) and incubated 180 minutes before High-Mass MALDI analysis (red traces). For each sample, after cross-linking, the non-covalent complexes [antibody/antigen] were detected in the high-mass range.

The Conclusions

To see the graphic analysis,

A red amino acid corresponds to a cross-linked amino-acid of the antigen with the antibody. A green amino acid corresponds to an X-ray crystallography interacting amino acid. A yellow amino acid corresponds to both cross-linked and X-ray crystallography interacting amino acids between the antibodies and the antigens.


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