Proteins are essential to all biological systems. Proteins participate in numerous cellular processes by interacting with other proteins, other metabolites and membranes in a dynamic environment. Studying the structural and conformational properties of proteins in the solution phase is necessary to understand their protein folding and interaction dynamics. This research project focused on the development and application of hydrogen deuterium exchange mass spectrometry (HDX-MS) technology for studying the conformational dynamics of large multi-subunit protein systems. HDX-MS studies were conducted on representative proteins of two much researched protein families, namely Peroxiredoxins (Prxs) and Cullin Ring Ligases (CRLs). As part of this research we implemented tandem mass spectrometry in the data independent acquisition (MS[supserscript E]) mode for the HDX-MS analysis. We also used ion mobility as a second and orthogonal dimension of separation to overcome the spectral crowdedness. Peroxiredoxins are ubiquitous antioxidant enzymes present in many organisms. Their catalytic activity is regulated by redox dependent oligomerization and their sensitivity to overoxidation is related to the flexibility of the active site loop to undergo partial unfolding. In this research we conducted HDX-MS experiments for determining to what extent the flexibility of the active site loop governs the sensitivity of peroxiredoxins to overoxidation. As example of a robust peroxiredoxin we studied initially the conformational properties of Salmonella typhimurium AhpC wild-type protein by HDX-MS. Subsequently, we conducted comparative HDX-MS analysis on the reduced form of the wild-type protein, and two single point mutants, T77V, and T77I, with the objective to decipher to what extent the stability of the dimer-dimer (A)interface affects the conformational dynamics of the active site loop. Differential HDX-MS results of the wild-type, disulfide reduced wild-type protein have exhibited a decrease in the motility of the active site loop and the C-terminal end of the protein upon disulfide reduction. The Thr77 single point mutation by valine enhanced the dimer-dimer interaction thereby stabilizing the decamer interface and increasing the motility of the active site loop. Whereas, the substitution of T77 by isoleucine increased the motility of the interfacial region which forms the dimer-dimer interface thereby promoting the dissociation of the decamer to dimers. A technically more advanced HDX-MS experimental setup was used to study the exchange-in properties of two robust peroxiredoxins, namely the wild-type StAhpC and the C46S mutant of StAhpC, which mimicks the reduced wild-type StAhpC, in comparison to human Prx2, a peroxiredoxin which is considered as sensitive to overoxidation. When differential deuterium uptake of wild-type StAhpC, C46S mutant StAhpC were compared, increased conformational rigidity was observed in the C46S mutant protein compared to the wild-type Prx. The peptide with highest deuterium incorporation levels in the human Prx2 is much lower compared to the bacterial wild type and C46S mutant Prxs. These comparative HDX-MS studies have fostered our understanding of the underlying conformational dynamics that lead to robust and sensitive Prxs. The second protein system that was studied was a representative of the Cullin Ring Ligases (CRLs), the largest family of RING-type E3 ligases that catalyze ubiquitylation of substrates. Protein ubiquitination is a post-translational modification that regulates several important biological processes in eukaryotic cells. It involves a three enzyme enzymatic cascade consisting of an ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligases (E3). In this study focus was directed toward the Cullin scaffold protein, which adopts an elongated structure that allows substrate receptor binding at the N-terminal domain (NTD) via adaptor proteins. Its C-terminal domain (CTD) binds to E2-ubiquitin through the RBX ring subdomain. Covalent attachment of the ubiquitin-like protein Nedd8 to the conserved lysine residue of the CTD stimulates the transfer of ubiquitin to substrate proteins thereby promoting ubiquitination. The HDX-MS studies of CUL1-RBX1 protein and its neddylated form highlighted that neddylation induces significant flexibility in the conformational dynamics of the CUL1 and RBX1 protein. The HDX-MS results support a mechanistic model in which conformational flexibility in the C-terminal domain of CUL1 and a concomitant opening of the RBX1 protein is necessary to allow the ubiquitin-bound E2 to be placed in close proximity to the protein substrates thereby facilitating the CRL activity.