D. J. Douglas

Collisional focusing effects in radio frequency quadrupoles

The transmission of ions through a conventional two-dimensional radiofrequency-only (rf) quadrupole has been studied for comparatively high operating pressures between 5 × 10−4 and 1 × 10−2 torr. Measurements of signals from mass-resolved analyte ions and total ion currents show that, provided the initial injection ion energy is low (1–30 eV), the ion transmission observed through a small aperture at the exit of the rf quadrupole first increases as the gas pressure increases, reaching a maximum at − 8 × 10−3 torr before decreasing at higher pressures. This is in direct contrast to the expectations of classical scattering. This “collisional focusing” appears to be analogous to effects seen in three-dimensional ion traps. The collisional focusing increases with the mass of the ion (not mass-to-charge ratio) for masses up to at least 16,950 u. The collisional focusing of the ions is found to be accompanied by significant losses of axial kinetic energy. A Monte Carlo simulation of the energy loss process is reported that can provide agreement with the observed losses for reasonable collision cross-sections. The results suggest that operation of rf quadrupoles at relatively high pressure may find practical application in sampling ions from high (e.g., atmospheric) pressure ion sources.

Collision cross sections for protein ions.

A method for the determination of cross sections for gas-phase protein ions, based on the energy loss of ions as they pass through a collision gas, is described. A simple model relates the energy loss to the number of collisions and hence the cross section. Results from a Monte Carlo model that support the validity of this approach are described. Experimental cross sections are reported for motilin, ubiquitin, cytochrome c, myoglobm, and bovine serum albumin. Cross sections range from approximately 800 Å2 for motilin to approximately 14,000 Å2 for bovine serum albumin and generally increase with the number of charges on the ion. Cytochrome c ions from aqueous solution show somewhat smaller cross sections than ions formed from solutions of higher organic content, suggesting that the gas-phase ions may retain some memory of their solution conformation.

A new linear ion trap time-of-flight system with tandem mass spectrometry capabilities

A new hybrid linear ion trap time-of-flight mass spectrometer has been constructed and the first results for tandem-in-time MS/MS in a 2-dimensional Paul trap are presented. The instrument consists of an electrospray ionization source and two radio frequency (RF) only quadrupoles operating at a pressure of 7 mTorr N2 which are coupled orthogonally to a linear TOFMS. Ions are trapped in an RF-only quadrupole through the application of timed stopping potentials on the entrance and exit apertures. Through the coupling of an auxiliary waveform generator on one pair of the ion trap rods a small dipolar excitation voltage is superimposed on the quadrupolar electric field. This voltage is used for precursor isolation via broadband excitation and collision induced dissociation through mass selective resonant excitation. Instrumental performance and choice of operating parameters are demonstrated by recording fragment spectra for +3 ions of renin substrate and +1 ions of reserpine. TOFMS mass resolution (m/Δm)FWHM is as high as 740 at m/z = 609. Fragmentation efficiency is greater than 50% and excitation mass resolution is 60–75.

Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry1997, 36, 12296–12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.

Matrix methods for the calculation of stability diagrams in quadrupole mass spectrometry

The theory of the computer calculation of the stability of ion motion in periodic quadrupole fields is considered. A matrix approach for the numerical solution of the Hill equation and examples of calculations of stability diagrams are described. The advantage of this method is that it can be used for any periodic waveform. The stability diagrams with periodic rectangular waveform voltages are calculated with this approach. Calculations of the conventional stability diagram of the 3-D ion trap and the first six regions of stability of a mass filter with this method are presented. The stability of the ion motion for the case of a trapping voltage with two or more frequencies is also discussed. It is shown that quadrupole excitation with the rational angular frequency ω = NΩ/P (where N, P are integers and Ω is the angular frequency of the trapping field) leads to splitting of the stability diagram along iso-β lines. Each stable region of the unperturbed diagram splits into P stable bands. The widths of the unstable resonance lines depend on the amplitude of the auxiliary voltage and the frequency. With a low auxiliary frequency splitting of the stability diagram is greater near the boundaries of the unperturbed diagram. It is also shown that amplitude modulation of the trapping RF voltage by an auxiliary signal is equivalent to quadrupole excitation with three frequencies. The effect of modulation by a rational frequency is similar to the case of quadrupole excitation, although splitting of the stability diagram differs to some extent. The methods and results of these calculations will be useful for studies of higher stability regions, resonant excitation, and non-sinusoidal trapping voltages.

Dissociation of Heme from Myoglobin and Cytochrome b5: Comparison of Behavior in Solution and the Gas Phase†,‡

The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme-protein complexes in the orifice-skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabilized in solution but stabilized in the gas-phase ions. These results suggest that on the millisecond time scale of the ES-MS experiment, the gas-phase protein ion may retain much of the structure of the protein in solution, at least for those residues surrounding the heme group. Furthermore, the ability of ES-MS to detect relatively subtle differences in protein-small molecule complex stability demonstrated in this work suggests that this technique may be a convenient, sensitive, and generally useful strategy for physical characterization of such complexes.

Applications of collision dynamics in quadrupole mass spectrometry

Some applications of collision dynamics in the field of quadrupole mass spectrometry are presented. Previous data on the collision induced dissociation of ions in triple quadrupole mass spectrometers is reviewed. A new method to calculate the internal energy distribution of activated ions directly from the increase in the cross section for dissociation with center of mass energy is presented. This method, although approximate, demonstrates explicitly the high efficiency of transfer of translational to internal energy of organic ions. It is argued that at eV center of mass energies, collisions between protein ions and neutrals such as Ar are expected to be highly inelastic. The discovery and application of collisional cooling in radio frequency quadrupoles is reviewed. Some previously unpresented data on fragment ion energies in triple quadrupole tandem mass spectrometry are shown that demonstrate directly the loss of kinetic energy of fragment ions in the cooling process. The development of the energy loss method to measure collision cross sections of protein ions in triple quadrupole instruments is reviewed along with a new discussion of the effects of inelastic collisions in these experiments and related ion mobility experiments.

The methanol-induced conformational transitions of β-lactoglobulin, cytochrome c, and ubiquitin at low pH: A study by electrospray ionization mass spectrometry

The methanol-induced conformational transitions under acidic conditions for β-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with β-sheets, α-helices, and both α-helices and β-sheets, respectively, are studied under equilibrium conditions by electrospray ionization mass spectrometry (ESI-MS). The folding states of proteins in solution are monitored by the charge state distributions that they produce during ESI and by hydrogen/deuterium (H/D) exchange followed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induced α-helix structure. Intermediate states formed at about 35% methanol concentration are found to give bimodal charge state distributions. The same rate of H/D exchange is shown by the two contributions to the bimodal distributions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchange of proteins followed by ESI-MS shows that helical denatured states, populated at around 50% methanol concentration, transform into more protected structures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins.

Reaction of human myoglobin and H2O2. Involvement of a thiyl radical produced at cysteine 110.

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2(protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO⋅) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g ∼ 2.036) was detected after decay of Trp-OO⋅ that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios ≤1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was detected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios ≥1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2demonstrated the formation of a homodimer (mass of 34,107 ± 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.

Excitation frequencies of ions confined in a quadrupole field with quadrupole excitation

Resonant quadrupole excitation of ions confined in a radio frequency quadrupole field with angular frequency Ω by an excitation signal with angular frequency ω has been investigated theoretically. It is shown that the spectrum of excitation frequencies has considerable structure which corresponds to different orders of excitation. The resonance condition for orders K = 1,2,3, … in the general case has been obtained as ωn(K) = (Ω/K) |n + β|, − ∞ < n < ∞, where K is the order of the resonance and β and n determine the unperturbed oscillation frequencies. Resonance curves for ion oscillations with different stability parameters β = 0.1, 0.5, and 0.9 have been constructed by means of direct numerical solution of the equations of motion. The trajectories of ion motion under resonant excitation of different orders have been investigated. For orders K of two and higher, the ion motion shows a beat character with an overall increase of amplitude with time. The stability diagram for ion motion in a mass filter in the presence of quadrupole excitation has been constructed.