Electrospray mass spectrometry as a method for studying the high pressure denaturation of proteins
Résumé
High pressure denaturation of proteins can provide important information concerning their folding and function. These studies require expensive and complicated equipment. In this paper we present a new convenient method for studying high-pressure denaturation of proteins combining deuterium-hydrogen exchange and electrospray mass spectrometry. Application of various values of pressure causes different degrees of protein unfolding resulting in molecules with a different number of protons available for exchange with deuterons. After decompression a protein refolds and a certain number of deuterons is trapped within the hydrophobic core of a refolded protein. Redissolving the deuterated protein in an aqueous buffer initiates the D/H exchange of amides located on the protein surface only, which can be monitored under atmospheric pressure by mass spectrometry. Depending on the degree of deuteration after high pressure treatment, the D/H exchange kinetics are different and indicate how many deuterons were trapped in the protein after refolding. The dependence of this number on pressure gives information on the denaturation state of a protein. The distribution of deuterium along the sequence of a high- pressure-denatured protein was studied the electron capture dissociation (ECD) on a Fourier-transform mass spectrometer, enabling the monitoring of high pressure denaturation with single amino acid resolution.
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