Abstract
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an important tool for measuring and monitoring protein structure. A bottom-up approach to HDX-MS provides peptide level deuterium uptake values and a more refined localization of deuterium incorporation compared with global HDX-MS measurements. The degree of localization provided by HDX-MS is proportional to the number of peptides that can be identified and monitored across an exchange experiment. Ion mobility spectrometry (IMS) has been shown to improve MS-based peptide analysis of biological samples through increased separation capacity. The integration of IMS within HDX-MS workflows has been commercialized but presently its adoption has not been widespread. The potential benefits of IMS, therefore, have not yet been fully explored. We herein describe a comprehensive evaluation of traveling wave ion mobility integrated within an online-HDX-MS system and present the first reported example of UDMSE acquisition for HDX analysis. Instrument settings required for optimal peptide identifications are described and the effects of detector saturation due to peak compression are discussed. A model system is utilized to confirm the comparability of HDX-IM-MS and HDX-MS uptake values prior to an evaluation of the benefits of IMS at increasing sample complexity. Interestingly, MS and IM-MS acquisitions were found to identify distinct populations of peptides that were unique to the respective methods, a property that can be utilized to increase the spatial resolution of HDX-MS experiments by >60%.Graphical ᅟ
Highlights
B etter characterization of higher order protein structure which encompasses secondary, tertiary and quaternary structures, is an important element to aid the understanding of the link between protein structure and biological function
These conditions are low pH and low temperatures with short analytical timeframes typically used. It is for this reason that protein digestion in local Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments is performed with an acid-tolerant enzyme prior to analysis by liquid chromatography mass spectrometry (LC-MS)
An initial assessment of the implementation of ion mobility within a HDX-MS workflow was performed on recombinant human growth hormone. Recombinant human growth hormone (rhGH) is a pituitary hormone that has been previously used in our lab as a model system for optimizing and assessing HDX-MS reproducibility and sensitivity
Summary
B etter characterization of higher order protein structure which encompasses secondary, tertiary and quaternary structures, is an important element to aid the understanding of the link between protein structure and biological function. Cryar et al.: HDX-IM-MS: a Systematic Evaluation nature of the HDX process a major technical caveat to both of these experiments is the need for all steps post-exchange to be performed at conditions which best quench the exchange process thereby improving the ability of the method to detect small conformational differences [12, 13] These conditions are low pH and low temperatures with short analytical timeframes typically used. It is for this reason that protein digestion in local HDX-MS experiments is performed with an acid-tolerant enzyme prior to analysis by liquid chromatography mass spectrometry (LC-MS). Pepsin is the favoured enzyme, due to its high activity at low pH and low cleavage site specificity
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