Analysis of “difficult” proteins by microfluidic-based hydrogen/deuterium exchange

Many proteins important in biology or disease are not amenable to traditional methods for structural analysis as they cannot be isolated in sufficient amounts nor studied directly in their native environment.

Alternative techniques are needed to understand the conformation and interactions of such difficult proteins and further support the on-going shift to protein-based ‘biopharmaceuticals’ in the pharmaceutical industry.

One promising technique to meet this challenge is the use of mass spectrometry to measure the hydrogen/deuterium exchange of proteins (HDX-MS). However, HDX-MS suffers from significant limitations to realize its full potential and applicability.

HDXchip aims to develop innovative microchips to revolutionize the HDX-MS method and enable analysis of difficult protein systems of unprecedented complexity.

HDXchip will create “HDX-MS 2.0” technology that integrates on-chip sample treatment and separation to overcome major limitations of the conventional method, including virtually eliminating unwanted deuterium loss (back-exchange). The key to fabrication of HDXchips lie in the fact that all elements will be made of thiol-ene polymers.

These polymers are not only used to make the chip itself in a cheap and simple manner, but also to fabricate high surface area functionalities for fast sample treatment (incl. enzymatic reactions) and separation at unprecedented efficiency.

HDXchip will enable step-change improvements in popular HDX-MS applications, like analysis of the conformation and interactions of purified proteins and also open up for exciting and hitherto unfeasible applications such as analysis of proteins inside whole cells and proteins in their native lipid membranes.

Hence, HDXchip could enable breakthrough insights into the conformation of difficult protein systems both in isolation and in their native environment, and dramatically improve our understanding of the function of such proteins and their potential to be targeted by drugs.