Protein Microarrays for High Throughput Hydrogen/Deuterium Exchange Monitored by FTIR Imaging

Area of Science:

• Biochemistry
• Biophysics
• Analytical Chemistry

Background:

• Proteins are a rapidly expanding class of therapeutics, but their inherent instability can lead to structural alterations.
• Changes in protein structure can compromise drug efficacy and safety, necessitating careful evaluation.
• Hydrogen deuterium exchange (HDX) is a valuable technique for assessing protein structure and dynamics, often monitored by mass spectrometry.

Purpose of the Study:

• To develop a high-throughput method for evaluating protein structure alterations using HDX.
• To combine protein microarrays with Fourier transform infrared (FTIR) spectroscopy for rapid HDX measurements.
• To enable real-time monitoring of HDX and automated data analysis.

Main Methods:

• Utilized protein microarrays on BaF2 slides for simultaneous HDX measurements.
• Employed FTIR imaging to monitor HDX in real-time across approximately 96 protein spots.
• Developed an experimental setup allowing continuous flushing with 2H2O-saturated N2 gas.
• Applied inverse Laplace transform and exponential curve fitting for HDX data analysis.

Main Results:

• Achieved high-throughput HDX measurements with FTIR imaging of protein microarrays.
• Enabled real-time monitoring of HDX, avoiding back-exchange issues.
• Demonstrated the feasibility of quantitative sample comparison through HDX curve analysis.
• Showcased the automation of the entire analysis process for rapid results.

Conclusions:

• The combined protein microarray and HDX-FTIR imaging technique offers a powerful tool for assessing protein stability and structural integrity.
• This method allows for rapid, high-throughput, and quantitative analysis of protein structure dynamics.
• The developed approach has significant implications for the development and quality control of protein-based therapeutics.