HDX SL 530x245

HDX Solution – unlock insights in protein structure conformation

  • Are you looking for a way to identify structural hotspots within your potential biotherapeutic protein?

  • Are you searching for a solution for understanding structural conformational changes of your biotherapeutic during formulation or storage?

  • Do you want to understand more about the protein-ligand (drug/protein) interactions and their relationship to drug activity?

The Bruker HDX Solution enables you to answer these questions and unlock insights into protein structure conformation, delivering reliable end results - first time, every time.

The Challenges of Biotherapeutic Development

Researchers within (bio)pharmaceutical sciences are increasingly tasked with gaining a deeper knowledge and understanding of the effects interactions and stressors such as pH or temperature, have on the conformational structure of biotherapeutic proteins. This is a very challenging task and researchers often use a variety of analytical tools to study the higher order structure of the biotherapeutics, monitor protein/protein-drug interactions and perform epitope mapping. 

In recent years Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) has emerged as a valuable technique within the arsenal of analytical tools available for providing such insights into protein structure conformation. However, until recently HDX-MS was confined mostly to academic research laboratories, due to a lack of automation and limited data interpretation tools, as well as the need to have reliable monoisotopic mass assignments.

HDX Solution Providing Fast, Automated, Reliable, Quantitative Structural Insights

The Bruker HDX Solution combines the LEAP HDx PAL, with the market-leading Bruker maXis II ETD and state of the art software from Sierra Analytics (HDExaminer) for data analysis. The industry-leading ETD capabilities of Bruker’s maXis II ETD UHR-QTOF offers unparalleled sequence coverage for middle-down and top-down studies while maintaining access to your existing peptide mapping workflows. However, the evolution of HDX-MS from bottom-up strategies based upon pepsin-digestion toward middle-down and top-down studies utilizing the power of ETD fragmentation has the potential to revolutionize the way conformational studies are performed.

The Bruker HDX Solution tool kit delivers automated and reliable HDX-MS workflows with correct monoisotopic accurate-mass readout to fully explore quantitative results from H/D ratios. This enables structural insights about biomolecules to be accessed reliably and with greater certainty and confidence. 

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HDX Solution website protein structure

Benefits of Combining Automated HDX with UHR-QTOF MS Technology

Key components of the robotic setuo for automatic HX MS experiments

Due to the high level of specificity and sensitivity achieved in both MS and MS/MS the use of Ultra-High Resolution Quadrupole Time-of-Flight (UHR-QTOF) technology for biotherapeutic charaterization is well-established. The ability of UHR-QTOFs to provide correct monoisotopic accurate-mass readout whilst maintaining true isotopic patterns is necessary for achieving accurate, reliable H/D ratios enabling Researchers to fully explore the quantitative results from HDX-MS studies.

The ability to couple accurate monoisotopic peptide mass information with data obtained from innovative fragmentation approaches such as Electron Transfer Dissociation (ETD) on the UHR-QTOF platform, provides additional insights. No longer do we just know which peptide regions are affected by the perturbation but we can now pinpoint the changes occurring at the residue level.  

However, for HDX-MS to be accepted as a routine, reliable technology within the (bio)pharmaceutical sciences, the ability to readily interpret the data obtained and automate the entire HDX-MS time-course experiment from sample preparation through to LC-MS analysis is necessary.

The Bruker HDX Solution for Automated Structural Conformation Characterization

The Bruker HDX Solution is a complete toolkit combining the LEAP HDx PAL, UHPLC pump, the Bruker maXis II ETD and Sierra Analytics HDExaminer software. 

By utilizing the most advanced (MS) vendor independent HDX system, the LEAP HDx PAL, robustness and reliability are built-in as standard. The LEAP HDx PAL automates the HDX experimental workflow, schedules and performs labelled experiments, followed by quenching and injection under cold conditions.

High Quality Chromatographic and MS Data

Representative Heat Map produced in Sierra Analytics HDExaminer software

Chromatographic separation under UHPLC conditions followed by mass spectrometric detection using the Bruker maXis II with its sub-ppm mass accuracy, high sensitivity and isotopic fidelity guarantees, high quality data and ultimately confidence in the H/D ratios.

 

Market-leading ETD capabilities open-up a new era in HDX-MS experiments. The ability to enhance knowledge by probing at the residue level not just the peptide level potentially gives additional specificity and highlights potential hotspots more readily. Top-down and middle-down approaches are also possible and provide complementary data to that obtained from traditional bottom-up pepsin-digestion based HDX-MS experiments.

 

Visualization and Insights

The information and insights gained following data analysis can be used to probe protein structure conformational changes in the presence of a perturbation.  

 

The regions responsible for the changes can be readily visualized by linking to crystal structure visualization programs such as PyMol.

Crystal structure representations of the protein HtpG undergoing undergoing structural changes in the presence of ATP based on data from HDX MS experiments

Innovative, Intelligent Sample Scheduling

One of the key features in bringing HDX-MS into the age of automation and into (bio)pharmaceutical laboratories is the innovative scheduling software, Chronos, included with the LEAP HDx PAL.

 

This software maximizes the chromatographic throughput and hence lab productivity by the intelligent organization and scheduling of the sample preparation steps including labelling, incubation, digestion and quenching.

Optimised Scheduling afforded by the Chronos software provided with the LEAP H D X PAL

The Most-widely Used HDX-MS Data Interpretation Software


The ability to interpret and analyze the mass spectral data providing a comprehensive picture of the deuteration uptake behaviour of a protein is provided by Sierra Analytics HDExaminer software. This is the most-widely used vendor-neutral solution available on the market and has a high degree of functionality and is easy-to-use.

The HDX Workflow

HDX-MS experiments are often a series of time-course experiments performed under at least 2 different conditions e.g. at 2 different pH levels, or at 2 different temperatures, or with and without an additive. 

 

Once the conditions for the series of experiments have been ascertained, the information is transferred to the Chronos scheduling software for automatic calculation of the pre-incubation, labelling, quenching, digestion and separation times. 

Flowchart representing the tasks performed automatically by the HDX Solution

Once everything is set-up and the acquisition started the system runs automatically preparing, injecting and analysing all the samples in the series.  After the mass spectrometric data has been collected this is uploaded to the HDExaminer software for calculation of the H/D ratios, generation of the deuteration uptake plots and production of heat maps. These data can then be exported to a 3-D crystal structure program such as PyMol for a 3-D visualization of the regions affected by the change being studied.
 

HDX Solution Workflow (click to enlarge):

HDX Solution workflow1
HDX Solution workflow2

Bruker Daltonics services are performed by factory trained and certified engineers. DIN EN ISO 9001:2000 requirements are fulfilled. 

 

Available services include:

  • Help desk service for a faster access to our support team, to your service order follow up and for general information
  • Planned maintenance, according to your requirement
  • Telephone support from highly-skilled troubleshooting professionals, to isolate and resolve hard- and software problems
  • Web-based remote instrument service for service diagnosis and applications support
  • Customer on-site repair and maintenance service
  • Spare parts availability typically over night or within a few working days worldwide
  • Compliance services for installation qualification, operational qualification / performance verification
  • Site planning and relocation
  • Advise to improve labs efficiency (consumable, method development & how to run the complete solution)