Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution

Hydrogen-deuterium exchange (HDX) coupled with electrospray ionization (ESI) in mass spectrometry (MS) has become an important method in the study of protein structure and dynamics. It provides an opportunity for obtaining conformational structures of protein motions in physiological conditions and offers unparalleled limits of detection. This method relies on the fact that the accessibility of exchangeable amide hydrogens of a protein to D2O induces H/D isotopic exchange. The exchangeable amide hydrogens that lack hydrogen bonding when exposed to solvent are exchanged rapidly. Protected amide hydrogens from tightly folded elements and have hydrogen bonding exchange slow and can be detected by mass spectrometry. This dissertation discusses basic fundamentals of both the conventional HDX ESI MS method that is called bottom-up approach that utilizes an enzymatic digestion and gas phase fragmentation method or a top-down approach to obtain hydrogen isotopic exchange at specific amide hydrogens.;Insulin, substance P and ubiquitin were used as model peptide and proteins to investigate the utility of these approaches. Bovine insulin was used to study the reduction of disulfide bonds using TECP as a reducing agent after the hydrogen isotopic reaction. The results show that the peptic fragments obtained from this method are smaller than those from the previous study without the reduction of disulfide bonds. Therefore, this approach offers more structural details and ease in identifying the peptic fragments. However, the drawback from back exchange reaction during the reduction time and chromatographic separation requires further study. Substance P was used to investigate gas phase fragmentation of the deuterated peptide by electron capture dissociation (ECD). The results show that this top-down approach is feasible for HDX MS method. The ECD fragmentation of the doubly charged ion of deuterium labeled substance P after chromatographic separation method that eliminated the rapid exchanged deuteriums at the side chains and termini produces less scrambling. Ubiquitin was used to further investigate gas phase fragmentation of deuterium labeled protein by ECD. The surprising results from direct infusion show that the higher charge states gain more deuteriums than the lower ones. On the contrary, the gas phase fragmentation of the deuterated protein from chromatographic separation that eliminated the rapid exchanged deuteriums show the same number of deuteriums for all charge states. Thus, the results from both experiments provide the labile exchanged deuterium locations. Human insulin was used to study protein conformational change in the presence of Congo red (CR) by pulsed HDX ESI MS. CR is known as an inhibitor of insulin fibrillization by binding at the C-terminus of the B-Chain of insulin. Remarkably, the ESI mass spectrum of labeled protein in the presence of CR shows bimodal pattern, suggesting that CR binds with the partial unfolded intermediate structure of the protein. Moreover, the local HDX resulted from peptic digestion show that the conformational structure of the insulin in the presence of CR is different from that of the native form.;Overall, the results presented highlight the ability of the methods to provide insight into the conformational changes of proteins in solution

Fast Determination of Human Insulin and Its Deamidation Product by Electrospray Ionization Mass Spectrometry

Application of the electrospray ionization (ESI) technique in mass spectrometry (MS) has played an important role in analysis of biological molecules and polymers. The focus of this thesis is to assess and extend the application of this technique in the rapid determination of human insulin and its deamidation product. Quantitative assessment of deamidation of human insulin generally invokes relatively slow chromatographic analysis. Faster cost-effective analysis using mass spectrometry with a low-resolution analyzer is challenging because the deamidation product is only one Dalton heavier than the 5808-Da protein; low-level contamination has only a small effect on the isotope envelope. Exploiting the chemical differences between insulin and its deamidation product (6 carboxylic acids, vs 7 following deamidation), we have extended an application of an alternative MS method based on the relative intensities of the high charge states in negative ion electrospray, using isopropylamine (IPA) to enhance anion intensities. Source parameters were optimized for high sensitivity and stability of higher charge states. The intensities of the higher charge states were increased in proportion to the amount of the deamidation product present in the mixtures. From the various ratios of the deamidation product, the study shows that this method was able to determine the ratio of the deamidation product in one minute with LOD = 0.7 % deamidation

DNA methylation in Friedreich ataxia silences expression of frataxin isoform E

Epigenetic silencing in Friedreich ataxia (FRDA), induced by an expanded GAA triplet-repeat in intron 1 of the FXN gene, results in deficiency of the mitochondrial protein, frataxin. A lesser known extramitochondrial isoform of frataxin detected in erythrocytes, frataxin-E, is encoded via an alternate transcript (FXN-E) originating in intron 1 that lacks a mitochondrial targeting sequence. We show that FXN-E is deficient in FRDA, including in patient-derived cell lines, iPS-derived proprioceptive neurons, and tissues from a humanized mouse model. In a series of FRDA patients, deficiency of frataxin-E protein correlated with the length of the expanded GAA triplet-repeat, and with repeat-induced DNA hypermethylation that occurs in close proximity to the intronic origin of FXN-E. CRISPR-induced epimodification to mimic DNA hypermethylation seen in FRDA reproduced FXN-E transcriptional deficiency. Deficiency of frataxin E is a consequence of FRDA-specific epigenetic silencing, and therapeutic strategies may need to address this deficiency.SCOPUS: ar.jinfo:eu-repo/semantics/publishe