Abstract by Anton Berg
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a highly applicable analytical technique for measuring the conformational dynamics of proteins in solution. The technique can be used to investigate the dynamical properties of single proteins, protein-protein interactions, and biopharmaceuticals. In brief, exposure of proteins to deuterium rich solvent will induce the exchange of hydrogen for the heavier isotope deuterium in the protein. Analysis by liquid chromatography coupled to mass spectrometry allows for selective determination of the deuterium incorporation in the back-bone amides of the proteins, which can provide information on the conformational dynamics of the analyzed protein. One important limitation to the technique is the inherent deuterium to hydrogen back-exchange occurring during analysis caused by the reversible nature of the deuterium labeling.
The present PhD project aimed to improve the bottom-up HDX-MS workflow through the use of microfluidics. In general, microfluidics can decrease sample and solvent consumption in analytical chemistry. It is possible to integrate and automate multiple steps of analytical workflow in a single system with very little dead-volume. Furthermore, microfluidics can often provide a low-cost alternative to their conventional counterparts. In the context of HDX-MS, we furthermore aimed to take advantage of the low thermal mass of a microfluidic chip to facilitate sub-zero temperature HDX-MS workflows to limit deuterium to hydrogen backexchange.
In the first part of the project, we developed a thiol-ene microfluidic system capable of automated proteolysis, and trapping, desalting and separation of protein and peptide samples. An in-house made chip holder and cooling system enabled cooling of spatially defined parts of the microfluidic chip to sub-zero temperatures and interfaced the chip with chromatographic pumps and a mass spectrometer. The chip enabled global and local HDX-MS analysis of model peptides and proteins with low back-exchange compared to a conventional setup for HDX-MS.
In the second part of the project, we provided the first comprehensive review on the subject of sub-zero temperature HDX-MS. Despite more than 20 scientific publications in which subzero temperature HDX-MS workflows were used, surprisingly little literature on the challenges and limitations of designing and using such workflows is currently available. In our review, we covered how the organic solvents needed to cool workflows below 0°C influence every step of HDX-MS workflows, including proteolysis, chromatographic separation, and the back-exchange kinetics. Furthermore, we covered hardware limitations related to the use of organic solvents.
In summary, the present PhD project has demonstrated the application of sub-zero temperature workflows for HDX-MS analysis of proteins for decreasing the back-exchange occurring during analysis. We have demonstrated the potential of low-cost microfluidic systems as an alternative to conventional HDX-MS equipment, and we have provided the first comprehensive review on sub-zero temperature HDX-MS.