Victor A. Mikhailov

Detergent-free mass spectrometry of membrane protein complexes

We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides.

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.

Infrared spectroscopy of Li(NH3)n clusters for n=4–7

Infrared spectra of Li(NH3)(n) clusters as a function of size are reported for the first time. Spectra have been recorded in the N-H stretching region for n=4-->7 using a mass-selective photodissociation technique. For the n=4 cluster, three distinct IR absorption bands are seen over a relatively narrow region, whereas the larger clusters yield additional features at higher frequencies. Ab initio calculations have been carried out in support of these experiments for the specific cases of n=4 and 5 for various isomers of these clusters. The bands observed in the spectrum for Li(NH3)(4) can all be attributed to N-H stretching vibrations from solvent molecules in the first solvation shell. The appearance of higher frequency N-H stretching bands for n > or =5 is assigned to the presence of ammonia molecules located in a second solvent shell. These data provide strong support for previous suggestions, based on gas phase photoionization measurements, that the first solvation shell for Li(NH3)(n) is complete at n=4. They are also consistent with neutron diffraction studies of concentrated lithium/liquid ammonia solutions, where Li(NH3)(4) is found to be the basic structural motif.

Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the “slow-heating” threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the “slow-heating” methods.

Activated ion electron capture dissociation (AI ECD) of proteins: Synchronization of infrared and electron irradiation with ion magnetron motion

Here, we show that to perform activated ion electron capture dissociation (AI-ECD) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a CO2 laser, it is necessary to synchronize both infrared irradiation and electron capture dissociation with ion magnetron motion. This requirement is essential for instruments in which the infrared laser is angled off-axis, such as the Thermo Finnigan LTQ FT. Generally, the electron irradiation time required for proteins is much shorter (ms) than that required for peptides (tens of ms), and the modulation of ECD, AI ECD, and infrared multiphoton dissociation (IRMPD) with ion magnetron motion is more pronounced. We have optimized AI ECD for ubiquitin, cytochrome c, and myoglobin; however the results can be extended to other proteins. We demonstrate that pre-ECD and post-ECD activation are physically different and display different kinetics. We also demonstrate how, by use of appropriate AI ECD time sequences and normalization, the kinetics of protein gas-phase refolding can be deconvoluted from the diffusion of the ion cloud and measured on the time scale longer than the period of ion magnetron motion.

Mass-selective soft-landing of protein assemblies with controlled landing energies.

Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the methods throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum.

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

Threshold photoelectron photoion coincidence spectroscopy and selected ion flow tube reactions of CHF3 : comparison of product branching ratios

The threshold photoelectron and threshold photoelectron photoion coincidence spectra of CHF3 in the range 13.5–24.5 eV have been recorded. Ion yields and branching ratios have been determined for the three fragments , and CF+. The mean kinetic energy releases into fragment ions involving either C–H or C–F bond cleavage have been measured, and compared with statistical and impulsive models. behaves in a non-statistical manner characteristic of the small-molecule limit, with the ground electronic state and low-lying excited states of being largely repulsive along the C–H and C–F coordinates, respectively. The rate coefficients and product ion branching ratios have been measured at 298 K in a selected ion flow tube for the reactions of CHF3 with a large number of gas-phase cations whose recombination energies span the range 6.3 through 21.6 eV. A comparison between the branching ratios from the two experiments, together with an analysis of the threshold photoelectron spectrum of CHF3, shows that long-range charge transfer probably occurs for the Ar+ and F+ atomic ions whose recombination energies lie above ca. 15 eV. Below this energy, the mechanism involves a combination of short-range charge transfer and chemical reactions involving a transition state intermediate. §In honour of Professor John Simons’ 70th birthday Festschrift.

Selected Ion Flow Tube Study of the Ion−Molecule Reactions of Monochloroethene, Trichloroethene, and Tetrachloroethene

Data for the rate coefficients and product cations of the reactions of a large number of atomic and small molecular cations with monochloroethene, trichloroethene, and tetrachloroethene in a selected ion flow tube at 298 K are reported. The recombination energy of the ions range from 6.27 (H3O(+)) through to 21.56 (Ne(+)) eV. Collisional rate coefficients are calculated by modified average dipole orientation theory and compared with experimental values. Thermochemistry and mass balance predict the most feasible neutral products. Together with previously reported results for the three isomers of dichloroethene ( Mikhailov, V. A. ; Parkes, M. A. ; Tuckett, R. P. ; Mayhew, C. A. J. Phys. Chem. A 2006, 110, 5760 ), the fragment ion branching ratios have been compared with those from threshold photoelectron photoion coincidence spectroscopy over the photon energy range of 9-22 eV to determine the importance or otherwise of long-range charge transfer. For ions with recombination energy in excess of the ionization energy of the chloroethene, charge transfer is energetically allowed. The similarity of the branching ratios from the two experiments suggest that long-range charge transfer is dominant. For ions with recombination energy less than the ionization energy, charge transfer is not allowed; chemical reaction can only occur following formation of an ion-molecule complex, where steric effects are more significant. The products that are now formed and their percentage yields are a complex interplay between the number and position of the chlorine atoms with respect to the C=C bond, where inductive and conjugation effects can be important.

Catenane versus ring: do both assemblies of CS2 hydrolase exhibit the same stability and catalytic activity?

Catenane structures, in which two or more rings are mechanically interlocked, have historically occupied one of the central places in the field of supramolecular chemistry. In contrast to synthetic small-molecule catenanes, examples of naturally-occurring catenanes are scarce. Here, we report thermodynamic and enzymatic studies on CS2 hydrolase, which exists in solution as a mixture of unique hexadecameric catenane and octameric ring forms. A combination of field-flow fractionation coupled to multi-angle laser light scattering (FFF-MALLS) and native mass spectrometric analyses revealed that the catenane form is converted into the ring form at elevated temperatures, whereas the ring does not assemble into the catenane under the same conditions. Measurements of the enzyme kinetics for the conversion of CS2 into COS and H2S showed that the ring form of CS2 hydrolase possesses higher enzyme efficiency (per monomer) than the catenane form, whereas the catenane form is overall more active (per assembly).