Don L. Rempel

Fast photochemical oxidation of protein footprints faster than protein unfolding.

Fast photochemical oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although oxidation via hydroxyl radical induces unfolding in proteins on a time scale of milliseconds or longer, FPOP is designed to limit (*)OH exposure to 1 micros or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three oxidation-sensitive proteins and found that the distribution of modification (oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification model. This model breaks down when a scavenger is not used and/or hydrogen peroxide is not removed following photolysis. The outcome verifies that FPOP occurs on a time scale faster than conformational changes in these proteins.

Relation of signal sensitivity and ion z-motion in cubic cells. Theory and implication for ion kinetic studies

Abstract The temporal effects of ion evaporation and ion relaxation along the z -axis (parallel to the magnetic field) on ion signal intensity are addressed in this paper. An understanding of these ion dynamical phenomena is important for quantifying ion abundance and measuring rates of ion/molecule reactions with Fourier transform mass spectrometry (FTMS) and trapped cell ion cyclotron resonance (ICR) mass spectrometry. A theory is developed to account for the effects of the z -axis oscillation amplitude of trapped ions on the ion signal. As the z -motions are damped and the ions relax to the center of the cell, ion signal increases. The qualitative features of the theory are tested for three broad classes of ions: unreactive, modestly reactive, and highly reactive in bimolecular, ion/molecule reactions. Ion compression, accomplished by steadily increasing the trap voltage, is introduced as a means of expediting z -axis relaxation so that temporal variations of signal due to relaxation phenomena can be minimized. Ion evaporation, on the other hand, can be avoided by using higher trapping fields in the cell. The implications of these phenomena for ion kinetic studies are discussed.

Space charge effects in Fourier Transform Mass Spectrometry. I. Electrons

Abstract Extensive efforts to develop the theory of Fourier Transform Mass Spectrometry (FTMS) have led to an understanding of the qualitative aspects of the technique and to quantitative predictions which have been experimentally verified. At the same time, a number of theoretical assertions have not yet been quantitatively tested. In particular, further quantitative comparisons between theoretical and experimental peak shapes, including determinations of height, width, area, and center position could prove useful to further development of the FTMS technique. All of these peak shape parameters are affected by the number of charged particles present in the analyser cell during FTMS analysis. Quantitative comparisons of experimental values of these parameters with the predictions of theory presumes accurate knowledge of the number of charged particles present in the analyser cell during the observation of ion cyclotron decay signals.

Mass-dependent z-excitation of ions in cubic traps used in FTMS

Abstract Fourier transform mass spectrometric ion excitation methods such as rapidly scanned radio frequencies (chirps) are intended to put momentum into the ion cyclotron mode only. However, momentum will also be transferred to the trapping oscillation mode of the ions, as is shown from both ion trajectory simulations and experimental data. Momentum transfer occurs because of a synchronization between ion motion and temporal changes of the z -component of the excitation field to produce a net average force toward the nearest trap plate. Because a stronger excitation field is associated with chirp excitation relative to rf bursts, the phenomenon is particularly apparent with chirps. To a rough approximation, the synchronization effect increases with decreasing mass, decreasing trap voltage, and increasing excitation amplitude. When the excitation amplitude is increased to accommodate higher magnetic fields, the effect is expected to increase also. The most troublesome manifestation of this effect is mass-dependent ejection of ions from the cell. Methods for reducing z -excitation are proposed.

Temperature Jump and Fast Photochemical Oxidation Probe Submillisecond Protein Folding

We report a new mass-spectrometry-based approach for studying protein-folding dynamics on the submillisecond time scale. The strategy couples a temperature jump with fast photochemical oxidation of proteins (FPOP), whereby folding/unfolding is followed by changes in oxidative modifications by OH radical reactions. Using a flow system containing the protein barstar as a model, we altered the proteins equilibrium conformation by applying the temperature jump and demonstrated that its reactivity with OH free radicals serves as a reporter of the conformational change. Furthermore, we found that the time-dependent increase in mass resulting from free-radical oxidation is a measure of the rate constant for the transition from the unfolded to the first intermediate state. This advance offers the promise that, when extended with mass-spectrometry-based proteomic analysis, the sites and kinetics of folding/unfolding can also be followed on the submillisecond time scale.

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ42 peptide, the proposed causative agent in Alzheimer’s disease. The approach incorporates pulsed hydrogen–deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen–deuterium exchange does not require modified Aβ42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.

An electrically compensated trap designed to eighth order for FT-ICR mass spectrometry.

We present the design, guided by theory to eighth order, and the first evaluation of a Fourier transform ion cyclotron resonance (FT-ICR) compensated trap. The purpose of the new trap is to reduce effects of the nonlinear components of the trapping electric field; those nonliner components introduce variations in the cyclotron frequency of an ion depending on its spatial position (its cyclotron and trapping mode amplitudes). This frequency spread leads to decreased mass resolving power and signal-to-noise. The reduction of the spread of cyclotron frequencies, as explicitly modeled in theory, serves as the basis for our design. The compensated trap shows improved signal-to-noise and at least a threefold increase in mass resolving power compared to the uncompensated trap at the same trapping voltage. Resolving powers (FWHH) as high as 1.7 × 107 for the [M + H]+ of vasopressin at m/z 1084.5 in a 7.0-tesla induction can be obtained when using trap compensation.

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

AbstractEpitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods. Figureᅟ

High pressure trapping in Fourier transform mass spectrometry: A radiofrequency-only-mode event

A new event for the Fourier transform mass spectrometry (FTMS) sequence is developed and demonstrated. During this event, called a radiofrequency (RF)-only mode event, the typical passive cubic trap of a Fourier transform mass spectrometer is made to operate as an active quadrupole ion trap. The transition between active and passive modes is developed so that ion loss as a consequence of the transition can be held to 15% or less. The adduct of the ion-molecule reaction of the 1,3-butadiene radical cation and methyl vinyl ether was detected during the Rf-only-mode event at a helium pressure of ∼1×10−3 torr even though this adduct is not detectable under standard FTMS operating conditions.

A scaling technique for studying the dynamics of high mass ions in fourier transform mass spectrometry: a preliminary report

Abstract The signals that have been observed for high mass biomolecule ions in Fourier transform mass spectrometry (FTMS) are characterized by low signal-to-noise and low resolution at low ion number whereas the spectral peaks for more moderate mass ions are good with high signal and resolution. We have proposed that the behavior of target high mass ions can be experimentally investigated with low mass ions at lower magnetic inductions. The justification of this proposal derives from the observation that solutions to the equations of motion for high and low mass ions are the same if the time parameter of the solution is scaled, and if the appropriate adjustments to the magnet induction are made. New results obtained for the molecular ion of benzene at lower magnet inductions support the relevance of scaled experiments for studying problems that occur at high mass. High mass ions modeled with the benzene molecular ion show good signal and resolution that degrades as the target mass is increased. Increasing the ion number improves the signal and resolution. Compensating the cubic trap improves signal and resolution for moderate ion numbers. This suggests a partial remedy for the problems at high mass.