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

Compound Characterization Services by HDX

Talk to Our Scientists

"*" indicates required fields

This field is for validation purposes and should be left unchanged.

Why Choose CovalX’s Compound Binding Characterization Services?

CovalX offers unique expertise in the field of compound or ligand binding analysis taking benefits of decades of experience in the field of Mass Spectrometry and Hydrogen Deuterium eXchange (HDX) analysis. With its team of experts and extensive experience in HDX, CovalX provides reliable results on a scheduled timeframe.

At CovalX, the compound binding analysis goes beyond the screening analysis provided by a typical ligand binding assay (LBA) providing higher confirmation and a greater level of data related to the binding. Typical ligand binding assays often utilize fluorescence, electrochemical or radioactive detection methods and are only able to detect presence or possible the extent of the complex formation. However, HDX-MS can localize the binding pocket and conformational changes caused by a compound binding to a target protein. This provides the richest level of data possible for ligand binding.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has emerged as an important tool for the development of small molecule therapeutics and biopharmaceuticals. HDX has been known as a useful tool towards ligand binding for over a couple of decades, with a review from 2014 showing several unique benefits of HDX-MS for the analysis of small molecules (HDX-MS guided drug discovery: small molecules and biopharmaceuticals – ScienceDirect).

HDX-MS is applicable for conformational or discontinuous interactions, as well as linear interaction sites. The analysis detects directly interaction sites of molecules, as well as, allosteric changes in the protein target. Additionaly, the interaction is analyzed in solution in native conditions, with the accuracy of mass spectrometry.

CovalX is using the latest mass spectrometry equipment, HDX-MS automation robotics and software to rapidly characterize the binding or conformational changes generated by compounds on their protein target.

CovalX Compound Characterization by HDX-MS Step by Step

HDX-MS has been utilized on a research scale for decades at the academic level and within larger pharmaceutical companies. With the recent advancements in automated robotics, sample handling, and data analysis software, HDX-MS can now be offered on a timescale and with reasonable sample consumption for more mainstream analysis. CovalX utilizes all of these latest software and hardware tools in its analysis services.

The HDX-MS analysis provides much more depth of data into the dynamics of the interaction and binding location when compared with other compound characterization 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 binding sites begins, CovalX first performs High-Mass MALDI analysis on the intact target protein. This initial screening utilizes CovalX exclusive High-Mass MALDI detections systems for intact interaction analysis to ensure that the HDX experiment is performed on characterized and controlled proteins.

This analysis step is unique to CovalX services. The goal of this analysis is to verify the integrity of the target protein and detect possible multimerization of the protein.

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 protein is complexed with the compound, this deuterium uptake rate is altered in the binding regions and/or regions that have undergone conformational changes. These differences can be measured accurately at various time points.

After initial screening, the unbound target protein and the protein-compound are diluted in a D2O solution.

At various time points, the Deuterium/Hydrogen exchange is quenched and the protein samples are then digested into peptides using appropriate proteolytic digestion.

The resulting peptides are then directly injected into a high resolution liquid chromatography-mass spec (LC-MS) system.

From the peptide mass fingerprints (PMF), deuterium exchange rate heat maps can be compared between the protein alone and the compound binding with the protein.

PMF protein

Compound Binding

Step 3: Data Analysis and Reporting

Finally, data is analyzed with full report generation using easy to understand HDX heat map descriptions and personal result presentation.

Although the HDX data is analyzing the deuterium exchange of the amino acids, the final data provided will have resolution of a peptide or less (few amino acids) depending upon the overlap of the peptides detected. Heat maps will be presented from each compound showing the regions of change detected.

HDX analysis can determine binding of linear, conformational or discontinuous binding upon a target protein as well as allosteric changes to the protein upon ligand binding. The detected regions of change are then highlighted in the final report using several 3D model simulations.


figure heat maps protein

Why is CovalX’s Compound Characterization Services the Best Option?

Expertise Experienced HDXMS team servicing the industry for more than 15 years.
Latest Equipment CovalX has the latest HDX robotics coupled to the mass spectrometer. CovalX owns fully dedicated HDX instrumentation.
Quality Work Advanced initial intact screening of proteins provides the most reliable rate of success.
Efficient processing is conducted using its design of experiment (DOE) software.
Affordable Competitive cost compared with other technologies.
Latest Software CovalX utilizes both third-party software in combination with proprietary home-built software for data analysis.
Fast Turnaround Four to six weeks delivery time depending on the size of the project

Timeline, Sample Requirements and Resolution

  • Sample Consumption: 450 µg of target protein.
  • Resolution down to 4-8 amino acids (depending on PMF).
  • Timeline: 4-6 Weeks depending on number of compounds.
  • Applicable for most therapeutic proteins.
  • No need for recombinant protein work, peptide synthesis, micro-array.
  • Cost: CovalX offers the lowest cost available for HDX analysis.

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.

Talk to Our Scientists

Our team is available to answer any of your questions about our products and services