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

## TL;DR

This paper introduces a high-throughput method using protein microarrays and FTIR imaging to monitor hydrogen/deuterium exchange, enabling rapid and accurate analysis of protein structure and stability.

## Contribution

The novel integration of protein microarrays with FTIR imaging allows real-time, high-throughput HDX analysis without back-exchange issues.

## Key findings

- The method enables simultaneous HDX analysis of approximately 96 protein spots with minimal sample volume.
- Real-time monitoring of HDX using FTIR imaging avoids back-exchange problems common in traditional methods.
- Quantitative comparison of protein samples is feasible through inverse Laplace transform and exponential curve fitting.

## Abstract

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF2 slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N2 gas saturated with 2H2O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm2. Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses.