Epitope Mapping Service
Antibody drug with four drug compounds linked to IgG immunoglobulin

Epitope Mapping Services by HDX-MS

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CovalX Epitope Mapping by HDX-MS Step by Step

HDX-MS has been utilized on a research scale for many years at the academic level and within larger pharmaceutical companies. More recently, with the advancement of automated robotics, sample handling, and data analysis software, HDX-MS can now be offered on a timescale and with sample consumptions reasonable for more mainstream analysis.

This technique provides low cost, lower sample consumption and quick turnaround times when compared with other conformational epitopes mapping techniques.

Moreover, CovalX first screens the intact proteins by high mass MALDI to ensure that no aggregation or target multimerization will affect the quality of the final results.

Step 1: Initial Sample Screening

Before the high-resolution analysis of the epitope begins, CovalX first performs High-Mass MALDI analysis on the intact antibody and antigen. This initial screening utilizes CovalX exclusive High-Mass MALDI detections systems to ensure that the HDX experiment is performed on characterized and controlled proteins.

This analysis is unique to CovalX services. The goal of this analysis is to verify:

  1. The integrity of both the antibody and the antigen
  2. The possible aggregation of the antibody
  3. The possible multimerization of the antigen
Initial sample screening graphic

Step 2: Hydrogen Deuterium eXchange (HDX-MS)

When diluted in heavy water (D2O), backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their hydrogen bonding and solvent accessibility. When the antigen is complexed with the antibody, this deuterium uptake rate is altered in the epitope regions. These differences can be measured accurately at various time points.

After initial screening, the unbound antigen and the antibody-antigen complex are diluted in a D2O solution

At various time points, the Deuterium/Hydrogen exchange is quenched at 0°C and pH 2.5

The protein samples are then digested into peptides using appropriate proteolytic digestion

The resulting peptides are then directly injected for microflow LC-MS/MS detection

From the peptide mass fingerprints (PMF), deuterium exchange rate heat maps can be compared between the antigen alone or the antigen with antibody binding

Hydrogen Deuterium eXchange (HDX-MS) figure

Step 3: Data Analysis and Report

Finally, data is analyzed using the latest software packages from multiple providers with full report generation using easy to understand HDX heat map descriptions and personal result presentation. Graphical characterization upon the PDB structure included as available.

HDX-MS Data Analysis and Report figure

CovalX Epitope Mapping Services by HDX-MS

CovalX offers unique expertise in the field of Epitope Mapping services by Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) based on more than a decade of specialization in mass spec characterization of protein interaction and protein complexes. CovalX unique technologies provide reliable results on a scheduled timeframe.

HDX MS HDXMS

CovalX epitope mapping service by HDX-MS allows linear and conformational epitope characterization with high resolution and highest success rate. CovalX is using the latest mass spectrometry equipment, HDX-MS automation robotics and software to rapidly characterize the binding sites using HDX-MS. Additionally, CovalX includes in all analysis an advanced intact analysis of the intact proteins and complexes (something unique to CovalX) to provide the highest rate of success for mapping projects, Finally, CovalX has technicians with combined decades of experience in optimizing and understanding HDX-MS workflows.

Why Choose CovalX’s Epitope Mapping Services by HDX-MS?

CovalX offers two techniques for conformational epitope mapping by mass spectrometry: Hydrogen Deuterium eXchange (HDX-MS) and Crosslinking Mass Spectrometry (XL-MS). Each of these technologies provides unique advantages.

Technique Sample handling is conducted using the latest automation robotics. Mass spectrometry and data analysis with the latest instrumentation and software
Expertise Experienced epitope mapping team servicing the industry for more than 15 years
Quality Work Unique mass spectrometric characterization of the intact proteins (aggregation, multimers, stoichiometry of the protein complexes) before starting the HDX-MS experiments. Our pre-HDX sample understanding provides the most reliable rate of success
Affordable Most competitive technique cost/resolution/timeline
Low Sample Consumption Only 300 μg of each antigen/antibody required
Fast Turnaround 4 weeks (or less with RUSH) delivery time

Timeline, Sample Requirements and Resolution

  • Sample Consumption: 300 ug of antigen and 300 ug of mAb
  • Resolution down to 1-5 amino acids (depending on PMF)
  • Timeline: 4 weeks of delivery time (less with RUSH)
  • Cost: CovalX offers the lowest cost available for HDX analysis

Notable Benefits with CovalX HDX-MS Service

  1. Initial characterization of intact protein and complex provides robust screening before beginning mapping experiments. This is unique to CovalX services
  2. Expertise: CovalX has over a decade of experience working analyzing protein complexes using mass spectrometry
  3. Latest Equipment: CovalX utilizes the latest mass spec, automation robotics and analysis software available.
  4. Applicable for conformational, discontinuous and linear epitopes as well as many other conformational protein studies
  5. The interaction is analyzed directly, by mass spectrometry

Other Services that CovalX HDX-MS provide

Biosimilar characterization
Higher-order structure
Conformational/Protein dynamics
Small molecules interaction (Compound binding analysis)
Protein-Protein interactions
Protein folding characterization

Download supporting materials

Hydrogen Deuterium eXchange Hydrogen Deuterium eXchange Mass Spectrometry Services (HDX-MS)


For example patents, publications, or comparison data please see our library or contact us directly.

An overview of epitope mapping technologies can be seen here.

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