Gas-phase metalloprotein complexes interrogated by ion mobility-mass spectrometry (original) (raw)
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The Analyst, 2011
The technique of ion mobility mass spectrometry (IM-MS) has become of increasing interest for rapid analysis of the conformations adopted by biological macromolecules. It is currently used routinely for analysis of explosives and illegal substances in airport and military security. In biophysical research, it can be used to determine the temperature dependent rotationally averaged collision cross section of gas-phase ions of proteins and nucleic acids along with their mass to charge ratios. Nanoelectrospray ionisation allows the gentle transfer of intact biomolecules from solutions in which the native form(s) are present, into the solvent free environment of a mass spectrometer. It is believed by many researchers that the experimental collision cross sections of these molecules should have some relationship to crystal structure coordinates. In this review we outline the different experimental methods that can be used to measure ion mobility; we also describe methods used to calculate collision cross sections from input coordinates. Following this survey of the methodological approaches to IM-MS, we then summarise IM-MS data published to date for some monomeric peptides and small soluble proteins, along with collision cross sections calculated from their crystal structure coordinates. Finally we consider the relationship between experimental gas-phase conformations and those adopted in crystals and give an outlook on the application of IM-MS as a tool for structural biology.
International Journal of Mass Spectrometry, 2018
In this study, a commercial uniform field drift tube ion mobility-mass spectrometer (IM-MS) was utilized to measure the gas-phase conformational populations of three well-studied proteins: ubiquitin (8566 Da), cytochrome c (12,359 Da), and myoglobin in both apo and holo forms (16,951 and 17,567 Da, respectively) in order to evaluate the use of this technology for broadscale structural proteomics applications. Proteins were electrosprayed from either acidic organic (pH ~3) or aqueous buffered (pH ~6.6) solution phase conditions, which generated a wide range of cation charge states corresponding to both extended (unfolded) and compact (folded) gas-phase conformational populations. Corresponding collision cross section (CCS) measurements were compiled for significant ion mobility peak features observed at each charge state in order to map the conformational landscapes of these proteins in both helium and nitrogen drift gases. It was observed that the conformational landscapes were similar in both drift gases, with differences being attributed primarily to ion heating during helium operation due to the necessity of operating the instrument with higher pressure differentials. Higher resolving powers were observed in nitrogen, which allowed for slightly better structural resolution of closely-spaced conformer populations. The instrumentation was found to be particularly adept at measuring low abundance conformers which are only present under gentle conditions which minimize ion heating. This work
Journal of The American Society for Mass Spectrometry, 2015
In the present work, the kinetic intermediates of holo-and apo-myoglobin were studied by correlating the ion-neutral collision cross section and time resolved H/D back exchange rate simultaneously in a trapped ion mobility spectrometer coupled to a mass spectrometer (HDX-TIMS-MS). The high mobility resolution of the TIMS cell permitted the observation of multiple IMS bands and complementary molecular dynamics simulations resulted in the assignment of candidate structures for each experimental condition studied (e.g., holo [M+8H] +8-[M+9H] +9 and apo [M+9H] +9-[M+19H] +19). Inspection of the kinetic intermediates suggests that the tertiary structure of apomyoglobin unfolds quickly upon the loss of the Fe protoporphyrin IX that stabilizes the interactions between the A, G, and H helices. In the absence of the porphyrin heme, the apomyoglobin unfolds to X n kinetic intermediates that vary in the extent of unfolding as a result of the observed charge state.
Analytical Chemistry, 2012
There has been a significant increase in the use of ion mobility mass spectrometry (IM-MS) to investigate conformations of proteins and protein complexes following electrospray ionization. Investigations which employ traveling wave ion mobility mass spectrometry (TW IM-MS) instrumentation rely on the use of calibrants to convert the arrival times of ions to collision cross sections (CCS) providing "hard numbers" of use to structural biology. It is common to use nitrogen as the buffer gas in TW IM-MS instruments and to calibrate by extrapolating from CCS measured in helium via drift tube (DT) IM-MS. In this work, both DT and TW IM-MS instruments are used to investigate the effects of different drift gases (helium, neon, nitrogen, and argon) on the transport of multiply charged ions of the protein myoglobin, frequently used as a standard in TW IM-MS studies. Irrespective of the drift gas used, recorded mass spectra are found to be highly similar. In contrast, the recorded arrival time distributions and the derived CCS differ greatly. At low charge states (7 ≤ z ≤ 11) where the protein is compact, the CCS scale with the polarizability of the gas; this is also the case for higher charge states (12 ≤ z ≤ 22) where the protein is more unfolded for the heavy gases (neon, argon, and nitrogen) but not the case for helium. This is here interpreted as a different conformational landscape being sampled by the lighter gas and potentially attributable to increased field heating by helium. Under nanoelectrospray ionization (nESI) conditions, where myoglobin is sprayed from an aqueous solution buffered to pH 6.8 with 20 mM ammonium acetate, in the DT IM-MS instrument, each buffer gas can yield a different arrival time distribution (ATD) for any given charge state.
Conformer selection of protein ions by ion mobility in a triple quadrupole mass spectrometer
Journal of the American Society for Mass Spectrometry, 1994
Electrospray mass spectra of multiply charged protein molecules show two distinct charge state distributions proposed to correspond to a more highly charged. open conformational form and a lower charged, folded form. Elastic collisions carried out in the radiofrequencyonly collision cell of a triple quadrupole mass spectrometer have dramatic effects on the appearance of the mass spectra. The different cross sectional areas of the conformers allow preferential selection of one charge state distribution over the other on the basis of ion mobility. Preferential selection is dependent on the nature and pressure of the target gas as well as the nature of the protein. In the case of positively charged horse heart arcomyoglobin * Note: The results reported in this article were obtained concurrently with, but independently of, the work recently reported by Covey and Douglas [1] and Feng and Konishi [2J, Address reprint requests to Prof.
International Journal of Mass Spectrometry and Ion Processes, 1997
Our high resolution ion mobility apparatus has been modified by attaching an electrospray source to perform measurements for biological molecules. While the greater resolving power permits the resolution of more conformations for BPTI and cytochrome c, the resolved features are generally much broader than expected for a single rigid conformation. A major advantage of the new experimental configuration is the much gentler introduction of ions into the drift tube, so that the observed gas phase conformations appear to more closely reflect those present in solution. For example, it is possible to distinguish between the native state of cytochrome c and the methanol-denatured form on the basis of the ion mobility measurements; the mass spectra alone are not sensitive enough to detect this change. Thus this approach may provide a quick and sensitive tool for probing the solution phase conformations of biological molecules.
International Journal of Mass Spectrometry, 2021
Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Journal of Visualized Experiments, 2010
Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (Ω).
Rapid Communications in Mass Spectrometry
The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques. Methods: The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. Results: Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N 2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. Conclusions: We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.