Book Description

Native mass spectrometry (MS) is a structural biology tool that probes proteins and protein complexes in the gas phase. In native MS, electrospray ionization (ESI) of protein samples are prepared in nondenaturing conditions and generate "native-like" protein ions, which retain noncovalent interactions observed in solution. Therefore, native MS is useful to provide information about the stoichiometry, topology, and ligand binding of protein complexes. Native MS coupled with ion mobility (IM) provides the momentum transfer collision cross section (omega), which is indicative of the size and shape of ions. Collision-induced unfolding (CIU) is an energy-dependent IM-MS technique that probes the unfolding of protein structures monitored by omega as a function of energy. This dissertation explores the utility of native MS and IM-MS analysis to study protein complexes. First, native MS analysis is used to investigate the stoichiometry of iron and sulfur in the endogenous Fe-S clusters binding to the F-box and leucine-rich protein 5 (FBXL5) and Skp1 from the Skp1-Cul1-Fbox (SCF) ubiquitin ligase in Chapter 2. The thermal activation in solution prior to ESI in combination with instrumental activation effectively elucidates the binding of [2Fe-2S] to FBXL5-Skp1 by reducing the presence of nonspecifically binding adducts (NSA). The effects of polarity on native-like avidin tetramers are characterized using native MS and IM-MS analysis in Chapter 3. The native MS of native-like avidin tetramer shows that the average charge state distribution is different between the cations and the anions. The native IM-MS results show that the omega of native-like avidin tetramer is similar regardless of the charge state and polarity. However, the CIU results display differences due to the charge state and polarity. In particular, the differences between the CIU results of 14+ and 14- avidin ions indicate that CIU analysis of the avidin ions is sensitive to solely polarity. In order to quantitatively compare the CIU results of 14+ and 14- avidin ions, a similarity score is developed. Similarity score analysis comparing the 14+ and 14- ions indicates that the largest difference in omega is observed at near 800 eV, while the greatest similarity is observed at low energy range (56 to ~400 eV). The utility of native MS and IM-MS analysis is explored to characterize antibodies (Abs) using two IgG2 samples (SIgG2 and AIgG2) in Chapter 4. These two samples are from the same subclass (IgG2), have the kappa light chain and were each purified from human myeloma plasma, but were from different manufacturing origins. Native MS results of the two samples indicate that the two Abs display vastly different apparent mass (SIgG2: ~154 kDa and AIgG2: 157, 159 kDa respectively) and the relative mass heterogeneity based on the peak width and shape. The IM-MS analysis demonstrates that the omega of the native-like Abs depends on the z, which contrasts from the omega of most proteins. The strong dependence of omega on z may be due to large differences in structural populations and/or the presence of flexible hinge region. The CIU analysis of SIgG2 and AIgG2 demonstrates that the greatest difference in omega between the two Abs is present at low energy with greater difference for anions than for cations. Overall, these results indicate that anions and low energy may preferentially provide significant differences when comparing similar proteins using native IM-MS and CIU analysis. Collision-induced unfolding (CIU) is increasingly used to study the effects of ligand binding to proteins and protein complexes. In chapter 5, a workflow is developed to more accurately assess the effects of ligand binding on the CIU stability. Mass spectra of the quadrupole-selected precursor ions at varying collision-energy display signals indicate that the precursor ions for CIU analysis is interfered by the presence of NSA despite extensive buffer exchange. Therefore, Srelative method is developed to determine the minimum collision-energy threshold at which all of the apparent NSA are removed from the initial m/z window of the precursor ions. More generally, Srelative may be used for quality control of CIU analysis.