Waters has turned what was a highly manual analytical process, where success was dependent on technical skill, into an integrated and automated system solution. This system leverages high-pressure nano- to microscale UPLC separations and high-resolution MSE mass spectrometry to answer important questions about biotherapeutic protein dynamics, conformation, and interactions, including:
- Epitope mapping
- Protein-drug binding
- Protein-protein interactions
- Aggregation
- Effects of mutation on protein conformation and dynamics
- Localization of conformational changes as a result of formulations and stability testing
Waters offers the only commercially-available complete system for HXMS studies, pairing our ACQUITY UPLC M-Class System with HDX Technology with a Waters mass spectrometer such as the Xevo G2-S QTof, and HDX application-specific software, DynamX.
THE SYSTEM
The unprecedented system-level design integrates all the steps necessary to accurately, reproducibly – and most of all, easily – perform protein conformation studies. The Waters UPLC HDX MS system removes the barriers to adopting high resolution LC and MS for routine studies of protein conformational changes, making HDX a core competency for your laboratory.
ACQUITY UPLC® M-Class with HDX Technology
A core element of the system is the HDX Manager, which is purpose-built to manage samples separated under higher operating pressures, at 0° C and with no compromise in deuterium recovery. Its improved chromamatographic resolution is paired with a state-of-the-art mass spectrometer such as the XevoG2-XS Qtof, SYNAPTG2-Si HDMS, SYNAPT XS, or Cyclic systems. The system also integrates on-line proteolysis with trapping capability and MSE technology for maximum sequence coverage of complex mixtures of peptic digest.
Enzymate™ Online Digestion Column
An immobilized pepsin column packed with mechanically strong BEH particles, the Enzymate BEH Pepsin Column (2.1 x 30 mm, 5 µm) is capable of operating at 15,000 psi, whose digestion efficiency is enhanced at both low and high digestion pressures.
HDX Phosphorylase B Check Standard
Used for digestion and separation experiments.
Mass Spectrometry
Ion mobility separation (IMS) is used in the UPLC/MS HDX workflow, providing additional, orthogonal separations to chromatography and mass dimensions. Overlapping interfering ions are successfully resolved by IMS and displayed in DynamX HDX Software.
DynamX Data Analysis Software v 3.0
In HDX studies, data are produced across multiple time points, multiple species, and with replicates. Curating this data manually is not time-efficient and requires expert interpretation. Interpretation of this data is a repetitive process that requires counting and measuring spectra.
DynamX Software is designed to systematically select spectra with predetermined criteria and measure the mass change of the deuterated form. The software automation was greatly simplified by utilizing the sharper peaks and better separation available with UPLC, and the comprehensive nature of the MSE detection. This automation, along with the capability to sort and display data, has been an important advance.
DynamX tracks all peptides that are reproducibly found in replicates, ensuring consistency in monitoring the deuterium exchange. The software also calculates the amount of deuteration and displays the results in convenient comparative views: uptake curves, a butterfly chart, and a difference chart. Data processing time is significantly reduced from months for manual processing to hours for automated processing.
DynamX software helps researchers assess possible conformational changes in their proteins quickly. It simplifies hydrogen-deuterium exchange interpretation by performing these tasks:
- Aggregates search results from ProteinLynx Global SERVER™ (PLGS) to generate a list of peptides for interrogation during the experiment.
- Processes mass spectrometer data files to search for ions belonging to each peptide.
- Determines deuterium uptake automatically.
- Aggregates data as a function of replication and experimental state.
- Visualizes data for easier interpretation.
- Allows interaction with data; easily navigate data and modify results if necessary.
PRINCIPLES OF HDX
Hydrogen deuterium exchange (HDX) MS works in many applications, including understanding how small-molecule therapeutics bind to protein targets, and performing epitope mapping. With this technology, deuterium in a solution replaces the amide hydrogen in a biomolecule’s backbone, and MS measures these changes. The results reveal information about protein structure.
Basic principles of hydrogen deuterium exchange
Amide hydrogen on the backbone of a biomolecule exchanges with deuterium in solution at different rates partly dependent on conformation. These changes can be measured by MS and therefore the degree of activity at various sites can be inferred. These locations can be identified and mapped to the primary sequence, which also allows easier visualization. Relative folding and dynamics can be determined from the different rates of uptake at different locations.
Learn more: "Performing Hydrogen/Deuterium Exchange with Mass Spectrometry" in BioPharm International.
Using UPLC/MSE provides better coverage
Using MSE means that a comprehensive dataset can be generated. All of the peptides are measured without bias by peptide signal intensity. This is an important feature for HDX studies where key information might be found in low-intensity peptides.
HDX by mass spectrometry opens new windows into the dynamics of biomolecules by providing information on the relative deuterium uptake of different conformations of a protein, or locations within a protein. Recent developments have made HDX with mass spectrometry a more accessible tool to study the dynamics of higher order structure (HOS) of biotherapeutics.
Industry and regulatory trends have very clearly moved towards an emphasis on conformation and the relationships between biomolecules. Therefore HDX is a key tool that is being rapidly adopted in more areas of biotherapeutic analysis.
