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

Project Information

HDXchip

Grant agreement ID: 101003052

DOI

10.3030/101003052

EC signature date 27 January 2021

Start date 1 September 2021

End date 30 June 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 999 752,00

EU contribution

€ 1 999 752,00

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 2 - HDXchip (Analysis of “difficult” proteins by microfluidic-based hydrogen/deuterium exchange)

Reporting period: 2023-03-01 to 2024-08-31

Summary of the context and overall objectives of the project

Proteins can be viewed as molecular machines inside a cell factory. In order to understand how these machines function or malfunction during disease, one needs to characterize their conformation and the parts of the machines that move (dynamics) or interact during work. The same is true for understanding the action of drugs at the molecular level as most small molecule drugs work by interacting with proteins. Furthermore, a rapidly growing proportion of drugs in development are protein-based and understanding the link between their conformation and their function is key to developing and exploiting the vast therapeutic potential of such biopharmaceuticals. Unfortunately, many proteins important in biology or disease are not amenable to traditional methods for analysis of their conformation or structure 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. 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).

HDXchip will enable step-change improvements in our ability to study the conformation and interactions of proteins including proteins in their native lipid membranes. This will dramatically improve our understanding of the function of such proteins and their potential to be targeted by drugs.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have completed the development of a prototype of HDXchip. This prototype (chip v3) allows loading of deuterium labelled protein samples, efficient online pepsin proteolysis and desalting and chromatographic separation prior to mass analysis. Furthermore, selective parts of this chip prototype can be cooled to sub-zero temperatures. This functional chip prototype v3 has now been used to perform the first microchip-enabled HDX-MS experiments on model peptides and model proteins. We are very excited to observe that using this prototype chip we can perform HDX-MS experiments with significantly reduced loss of deuterium label (back-exchange) compared to conventional HDX-MS equipment thus improving the sensitivity of the measurement. We are still in the process of optimization. Importantly, this chip prototype costs only about 20 EUR in manufacturing costs – which is considerably lower cost compared to conventional commercially-available HDX-MS equipment (costs at least 100.000 EUR). We thus anticipate that this chip technology is poised to make HDX-MS technology more accessible to the wider scientific community due to its reduced costs.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In HDXchip, we are developing a novel microfluidic-based analytical technology capable of fast analysis of the conformation of “difficult” proteins by measuring their hydrogen/deuterium exchange in solution using mass spectrometry (HDX-MS). We have so far succesfully developed a prototype of HDXchip which represents a significant improvement to state-of-the-art in terms of ease of manufacturing, cost and sensitivity (back-exchange). Using the HDXchip prototype, we have performed the first microchip-enabled HDX-MS experiments at sub-zero temperatures. Work is on-going to improve the developed HDXchip prototype - and add new on-chip functionalities. We are convinced that the developed HDXchip technology will enable step-change improvements in our ability to study the conformation and interactions of proteins and thus improve our understanding of the function of such proteins and their potential to be targeted by drugs. Our long-term VISION for HDXchip is to make second generation HDX-MS technology that can be used outside our lab and be commercialized in the future. HDXchip is thus more than a “microfluidic toy”, but has real-world potential to replace existing liquid chromatography-based HDX-MS technology. Also, HDXchip is readily adaptable to other popular analytical workflows that rely on sample treatment and LC-MS, e.g. quality control of biopharmaceuticals, proteomics and a wide range of small molecule analyses. Therefore, HDXchip has broad potential to solve analytical challenges across biology, chemistry and the life sciences.

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