Biomedical subjects

D J Douglas

Publications and source records attributed to D J Douglas.

At least 19 recordsLinked to original sources

PubMed2006

Linear quadrupoles with added hexapole fields.

Linear quadrupoles with added hexapole fields are described. The shifts in ion oscillation frequency caused by the addition of a hexapole field are calculated within the effective potential model. Methods to construct linear quadrupoles with added hexapole fields with exact electrode geometries and with round rods are discussed. A quadrupole with added hexapole field can be constructed with round rods by rotating two rods (say the y rods) towards an x rod. Computer simulations are used to investigate the possibility of mass analysis with quadrupoles with added hexapole fields. We find that a quadrupole with an added hexapole field in the range 2-12% can provide mass analysis provided the dc is applied with the correct polarity and value. When a rod set is constructed with round rods, other multipoles in the potential degrade the peak shape, resolution and transmission. The largest of these after the quadrupole and hexapole are a dipole and octopole term. With round rod sets, the peak shape can be improved by using different diameters for the x and y rod pairs to minimize the octopole term in the potential and by injecting ions at the field center where the dipole term is zero. Calculations of the boundaries of the stability diagram for this case show the boundaries move out, relative to those of a pure quadrupole field, but remain sharp.

Journal Article↗

PubMed2006

Coulomb effects in binding of heme in gas-phase ions of myoglobin.

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry. With negative ions, neutral heme is lost regardless of the charge state of Fe in solution. It is likely that the Fe(3+) is reduced to Fe(2+) in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed with ions formed from ferromyoglobin in solution, and positive heme loss with ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model potential with a long-range Coulomb repulsion and short-range attraction between the heme and protein.

Algorithms↗

PubMed2005

Ion excitation in a linear quadrupole ion trap with an added octopole field.

Modeling of ion motion and experimental investigations of ion excitation in a linear quadrupole trap with a 4% added octopole field are described. The results are compared with those obtained with a conventional round rod set. Motion in the effective potential of the rod set can explain many of the observed phenomena. The frequencies of ion oscillation in the x and y directions shift with amplitude in opposite directions as the amplitudes of oscillation increase. Excitation profiles for ion fragmentation become asymmetric and in some cases show bistable behavior where the amplitude of oscillation suddenly jumps between high and low values with very small changes in excitation frequency. Experiments show these effects. Ions are injected into a linear trap, stored, isolated, excited for MS/MS, and then mass analyzed in a time-of-flight mass analyzer. Frequency shifts between the x and y motions are observed, and in some cases asymmetric excitation profiles and bistable behavior are observed. Higher MS/MS efficiencies are expected when an octopole field is added. MS/MS efficiencies (N(2) collision gas) have been measured for a conventional quadrupole rod set and a linear ion trap with a 4% added octopole field. Efficiencies are chemical compound dependent, but when an octopole field is added, efficiencies can be substantially higher than with a conventional rod set, particularly at pressures of 1.4 x 10(-4) torr or less.

Ions↗

PubMed2003

Conformations of gas-phase lysozyme ions produced from two different solution conformations.

Near pH 2.0, lysozyme in water is in its native conformation, and in water/methanol (2/8) it adopts a helical denatured conformation (Kamatari et al. Protein Sci. 1998, 7, 681-688). Hydrogen/deuterium (H/D) exchange of lysozyme in solution confirms that it is partially unfolded at pH 2.0 in water/methanol (v/v = 2/8). With electrospray ionization (ESI) mass spectrometry (MS), lysozyme in water produces ions with charges +7 to +12, with the greatest intensity at +10, whereas lysozyme in water/methanol (2/8) produces ions with charges +6 to +12 with the greatest intensity at +7. Thus, lysozyme is an exception to the rule that a protein denatured in solution forms higher charge states than the same protein in its folded native conformations in solution. Because the same charge states are produced from these two solution conformations, a direct comparison of the properties of the gas-phase ions produced from two very different solution conformations is possible. The conformations of lysozyme ions in the gas phase were studied using cross section measurements and gas-phase H/D exchange. Similar cross sections and H/D exchange levels were observed for same-charge states of lysozyme ions formed from the native and helical denatured conformations in solution. Cross sections show that the ions have compact structures. Thus, disulfide-intact gaseous lysozyme ions generated from the denatured state in water/methanol (2/8) refold into compact structures in the gas phase on a time scale of milliseconds or less.

Animals↗

PubMed2003

Linear quadrupoles with added octopole fields.

Two methods of adding relatively small octopole fields to the main quadrupole field of quadrupoles and linear ion traps with cylindrical rods are investigated. The first, 'stretching' the quadrupole by moving two rods out from the axis, produces a combination of higher order fields with similar magnitudes in which the octopole field is not necessarily the greatest. The quadrupole field strength is changed significantly and a large potential appears on the axis. The second method uses rod pairs of different diameters. It adds octopole components of up to several percent while all other higher order fields remain small. An axis potential is also added, but it is only a few percent of the radio-frequency (RF) voltage and approximately equal to the strength of the octopole field. The axis potential can be removed by moving the larger rod pair out from the axis or applying unbalanced RF to the electrodes.

Journal Article↗

PubMed2003

Quadrupole mass filters with octopole fields.

The performance of quadrupole mass filters with added octopole fields in the range 2.0-4.0% has been investigated. The added fields are much greater than those normally added to conventional rod sets by mechanical tolerances or construction errors. Quadrupole rod sets with added octopole fields were constructed with round rods by making one pair of rods greater in diameter than the other pair. For positive ions, resolution at half height of only about 200 is possible if the negative direct current (dc) output of the quadrupole power supply is connected to the smaller rods. If the positive dc output of the quadrupole power supply is connected to the smaller rods, the resolution improves dramatically; a resolution at half height of 5800 has been observed with a rod set with 2.6% added octopole field. For negative ions the best resolution is obtained with the polarity of the dc reversed, i.e. with the negative dc applied to the smaller rods. These findings are unexpected in view of the literature that argues that to obtain high mass resolution with quadrupole mass filters, higher order multipoles must be kept as small as possible. Numerical simulations of peak shapes agree qualitatively with experiments. Simulation of the boundaries of the first stability region for positive ions shows that when the positive dc is applied to the smaller rods, the addition of a 2.0% octopole field causes the boundaries to shift slightly but the boundaries are well defined, and the tip of the stability region remains sharp. When the positive dc is applied to the larger rods, the boundaries of the stability region move out and become diffuse. For instruments that require a rod set that can be used both as a linear trap and a mass filter, these rod sets may offer improved trap performance while still being capable of providing conventional mass analysis.

Journal Article↗

PubMed2003

H/D exchange of gas phase bradykinin ions in a linear quadrupole ion trap.

The gas phase H/D exchange reaction of bradykinin ions, as well as fragment ions of bradykinin generated through collisions in an orifice skimmer region, have been studied with a linear quadrupole ion trap (LIT) reflectron time-of-flight (rTOF) mass spectrometer system. The reaction in the trap takes only tens of seconds at a pressure of few mTorr of D2O or CD3OD. The exchange rate and hydrogen exchange level are not sensitive to the trapping q value over a broad range, provided q is not close to the stability boundary (q = 0.908). The relative rates and hydrogen exchange levels of protonated and sodiated +1 and +2 ions are similar to those observed previously by others with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer system. The doubly and triply protonated ions show multimodal isotopic distributions, suggesting the presence of several different conformations. The y fragment ions show greater exchange rates and levels than a or b ions, and when water or ammonia is lost from the fragment ions, no exchange is observed.

Bradykinin↗

PubMed2002

Influence of the 6th and 10th spatial harmonics on the peak shape of a quadrupole mass filter with round rods.

The influence of the ratio of the rod radius, r, to field radius, r(0), on the peak shape for a linear quadrupole mass filter constructed with round rods has been investigated. The expansion of the potential in multipoles, phi(N),Phi(x, y) = sum(infinity)(N=0)A(N)phi(N)/r(N)(0) has been considered, and the peak shape and resolution have been determined by numerical calculation of ion trajectories in quadrupoles with different ratios, r/r(0). Geometries that make the dodecapole term (A(6)) zero (r/r(0) = 1.14511) do not give the best performance because the contribution of the 20-pole term, A(10), must be considered. The optimum ratio is r/r(0) approximately 1.13. With this ratio the dodecapole term (A(6)) is ca. 1 x 10(-3), but its effects are partially compensated by the A(10) term which has similar magnitude, but opposite sign.

Journal Article↗

PubMed2002

Multiple sprayer system for high-throughput electrospray ionization mass spectrometry.

A multiple sprayer electrospray ion source for high-throughput analysis is described. The ion source is comprised of multiple electrospray capillaries, each with an ion lens located near the tip. The electric potentials applied to the ion lenses are used to control the sprayers. The use of ion lenses eliminates the need for mechanical blocking devices to selectively enable or disable the sprayers, and results in a less expensive and more reliable set-up. Sprayers can be enabled or disabled within approximately 50-250 ms when the lens potentials are controlled manually. For simultaneous operation of multiple electrospray capillaries, it is advantageous to orient the capillaries so that the spray from each passes directly in front of the entrance aperture of the mass spectrometer.

Journal Article↗

PubMed2002

Hydrogen/deuterium exchange of myoglobin ions in a linear quadrupole ion trap.

The hydrogen/deuterium (H/D) exchange of gas-phase ions of holo- and apo-myoglobin has been studied by confining the ions in a linear quadrupole ion trap with D(2)O or CD(3)OD at a pressure of several mTorr. Apo-myoglobin ions were formed by collision-induced dissociation of holo-myoglobin ions between the orifice and skimmer of the ion sampling system. The exchange takes place on a time scale of seconds. Earlier cross section measurements have shown that holo-myoglobin ions can have more compact structures than apo-myoglobin. Despite this, both holo-myoglobin and apo-myoglobin in charge states +8 to +14 are found to exchange nearly the same number of hydrogens (ca. 103) in 4 s. It is possible the ions fold or unfold to new conformations on the much longer time scale of the exchange experiment compared with the cross section measurements.

Apoproteins↗

PubMed2002

Tandem mass spectrometry of protein-protein complexes: cytochrome c-cytochrome b5.

An improved method to interpret triple quadrupole MS/MS experiments of complexes of large ions is presented and applied to a study of the complex formed by the proteins cytochrome c and cytochrome b5. Modeling of the activation and dissociation process shows that most of the reaction occurs near the collision cell exit where ions have the highest internal energies. Experiments at different collision cell pressures or with different collision gases (Ne, Ar, Kr) are interpreted with a previously proposed collision model (Chen et al., Rapid Commun. Mass Spectrom. 1998, 12, 1003-1010) to calculate the internal energy added to ions to cause dissociation. Small but systematic differences under different experimental conditions are attributed to different times available for reaction. A method to correct for this is presented. Ne, Ar, and Kr are found to have similar energy transfer efficiencies. Complexes of cytochrome c and cytochrome b5 are detected in ESI mass spectra but with abundances less than expected from the solution equilibrium. Dissociation of the cytochrome c-cytochrome b5 complexes with charge k gives as the most abundant fragments, cytochrome b5(+3) and cytochrome c+(k-3). Adding charges to the complex destabilizes it. A series of cytochrome c variants with Lys residues thought to be involved in solution binding replaced by Ala showed no differences in the energy required to induce dissociation of the gas phase complex. The implications for the binding of the gas phase ions are inconclusive.

Animals↗

PubMed2002

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 omega = Nomega/P (where N, P are integers and omega is the angular frequency of the trapping field) leads to splitting of the stability diagram along iso-beta 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.

Journal Article↗

PubMed2002

An atmospheric pressure ion lens that improves nebulizer assisted electrospray ion sources.

An atmospheric pressure ion lens improves the performance and ease of use of a nebulizer assisted electrospray (ion spray) ion source. The lens is comprised of an oblong-shaped stainless steel ring attached to an external high voltage power supply. The lens is located near the tip of the conductive sprayer, and is maintained at a potential less than that of the sprayer. The ion lens improves the shape of the equipotential lines in the vicinity of the sprayer tip. This lens gives approximately a 2-fold reduction in the signal RSD, a 2-fold increase in the ion signal, an increase in the number of multiply charged ions, and a much broader range of usable sprayer positions.

Journal Article↗

PubMed2001

Reaction of human myoglobin and peroxynitrite: characterizing biomarkers for myoglobin-derived oxidative stress.

Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury.

Biomarkers↗

PubMed2001

Detection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible.

Animals↗

PubMed2001

Ion fragmentation in an electrospray ionization mass spectrometer interface with different gases.

Nitrogen, argon, and krypton are used as curtain gases in an electrospray ionization mass spectrometer in an attempt to study the effect of these gases on the extent of ion fragmentation between the orifice and the skimmer of the interface region. A previously published collision model predicts that the degree of ion fragmentation increases with increasing mass of the curtain gas. However, the fragmentation yields are found to be the opposite to that expected. It is believed that the reversed trend with argon and krypton is caused by condensation of the gases within the free jet expansion between the orifice and the skimmer. A condensation parameter can be used to predict the degree of clustering of gases within a free jet expansion. When the condensation parameter is minimized, the predicted trend of fragmentation with mass is observed.

Argon↗

PubMed2001

A combined linear ion trap time-of-flight system with improved performance and MS(n) capabilities.

A detailed description of a linear ion trap time-of-flight (TOF) mass spectrometer system, capable of sequential mass spectrometry (MS(n)), is given. Many improvements have been incorporated since the initial description of this system (Rapid Commun. Mass Spectrom. 1998; 12: 1463-1474). The pressure in the trap has been lowered from 7.0 to 1.8 mTorr, resulting in an increase in the mass resolution of ion excitation from 75 to 240. Use of the system for MS(3) is demonstrated. Dipole excitation of the n = 1 harmonic, instead of the n = 0 fundamental frequency of ion motion, is shown to have a higher frequency resolution, f/Deltaf, but lower mass resolution, m/Deltam. Both experiments and modeling demonstrate that at the lower pressure there is less collisional cooling of ions in the axial and radial directions of the trap. The efficiency of trapping is shown to be nearly 100% for periods up to 5 s. The demonstrated mass range for mass analysis has been extended to greater than m/z 16 250. To avoid the formation of adduct ions when trapping protein ions for extended times requires ultra-high vacuum cleanliness conditions, even though the trap operates in the mTorr-pressure range. Upgrading the TOF to a reflectron with higher quality ion optics results in an increase in the mass resolution of the TOF mass spectrometer to about 5000 at m/z 750.

Angiotensinogen↗

PubMed2001

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

The methanol-induced conformational transitions under acidic conditions for beta-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with beta-sheets, alpha-helices, and both alpha-helices and beta-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 alpha-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.

Cytochrome c Group↗