In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

rationaleMany different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in-droplet hydrogen-deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection.

methodsTwo vibrating capillary vibrating sharp-edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D

resultsIn-droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix-forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in-droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A-state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions.

conclusionThe deuterium uptake after in-droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.