Biomedical subjects

D J Douglas

Publications and source records attributed to D J Douglas.

At least 37 records · Page 2Linked to original sources

PubMed2001

Collision-induced dissociation of bradykinin ions in the interface region of an ESI-MS.

By applying different electric field strengths to the orifice-skimmer region of an electrospray ionization mass spectrometer, the rate of dissociation can be varied based on the amount of internal energy acquired by an ion through collisions with the curtain gas molecules. Both the Arrhenius equation and Rice-Ramsperger-Kassel (RRK) theory can be used to predict the rate of dissociation of internally excited molecules. A previously determined model for collision-induced dissociation is tested by comparison of predicted and experimentally observed orifice-skimmer potential differences for dissociation of ions. The rate of collision-induced dissociation of bradykinin ions is determined by monitoring the fragments produced in a mass spectrometer. The semi-quantitative model is found to yield effective predictions when accurate Arrhenius and RRK parameters are utilized.

Algorithms↗

PubMed2000

Methanol-induced conformations of myoglobin at pH 4.0.

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.

Animals↗

PubMed2000

An interface with a linear quadrupole ion guide for an electrospray-ion trap mass spectrometer system.

A new ion sampling interface for an electrospray ionization 3D ion trap mass spectrometer system is described. The interface uses linear rf quadrupoles as ion guides and ion traps to enhance the performance of the 3D trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. A resolution of ejection of up to 254 is demonstrated for protonated reserpine ions (m/z 609.3). A composite waveform with a notch in frequency space is used to eject a wide range of matrix ions and to isolate trace analyte ions in a linear quadrupole before ions are injected into the 3D trap. This is useful to overcome space charge problems in the 3D trap caused by excess matrix ions. For trace reserpine in a 500-fold molar excess of poly(propylene glycol) (PPG), it is demonstrated that the resolution and sensitivity of the 3D trap can be increased dramatically with ejection of the excess PPG matrix ions. In comparison to ejection of matrix ions in the 3D trap with a similar broad-band waveform, a 5-fold increase in sensitivity with a 7 times shorter acquisition time was achieved.

Journal Article↗

PubMed2000

Reaction of human myoglobin and nitric oxide. Heme iron or protein sulfhydryl (s) nitrosation dependence on the absence or presence of oxygen.

The amino acid sequence of human myoglobin (Mb) is similar to other mammalian Mb except for a unique cysteine residue at position 110 (Cys(110)). Anaerobic treatment of ferrous forms of wild-type human Mb, the C110A variant of human Mb or horse heart Mb, with either authentic NO or chemically derived NO in vitro yields heme-NO complexes as detected by electron paramagnetic resonance spectroscopy (EPR). By contrast, no EPR-detectable heme-NO complex was observed from the aerobic reactions of NO and either the ferric or oxy-Mb forms of wild-type human or horse heart myoglobins. Mass analyses of wild-type human Mb treated aerobically with NO indicated a mass increase of approximately 30 atomic mass units (i.e., NO/Mb = 1 mol/mol). Mass analyses of the corresponding apoprotein after heme removal showed that NO was associated with the apoprotein fraction. New electronic maxima were detected at A(333 nm) (epsilon = 3665 +/- 90 mol(-)(1) cm(-)(1); mean +/- S.D.) and A(545 nm) (epsilon = 44 +/- 3 mol(-)(1) cm(-)(1)) in solutions of S-nitrosated wild-type human Mb (similar to S-nitrosoglutathione). Importantly, the sulfhydryl S-H stretch vibration for Cys(110) measured by Fourier transform infrared (nu approximately 2552 cm(-)(1)) was absent for both holo- and apo- forms of the wild-type human protein after aerobic treatment of the protein with NO. Together, these data indicate that the reaction of wild-type human Mb and NO yields either heme-NO or a novel S-nitrosated protein dependent on the oxidation state of the heme iron and the presence or absence of dioxygen.

Animals↗

PubMed2000

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 H(2)O(2) (protein/H(2)O(2) = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO( small middle dot)) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g approximately 2. 036) was detected after decay of Trp-OO( small middle dot) that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-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 H(2)O(2) demonstrated 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 H(2)O(2) differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.

Animals↗

PubMed2000

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 omega by an excitation signal with angular frequency omega 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 omega n(K) = (omega/K) magnitude of n + beta, -infinity < n < infinity, where K is the order of the resonance and beta and n determine the unperturbed oscillation frequencies. Resonance curves for ion oscillations with different stability parameters beta = 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.

Algorithms↗

PubMed1998

Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry.

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.

Bacillus↗

PubMed1998

Conformation of native, reduced and [5-55]Ala bovine pancreatic trypsin inhibitor in the gas phase.

Collision cross sections have been measured for gas phase ions of native oxidized bovine pancreatic trypsin inhibitor (BPTI), native reduced BPTI and a mutant form of BPTI containing a single disulphide bond between residues 5 and 55 ([5-55]Ala BPTI). Cross sections for [5-55]Ala BPTI and reduced BPTI were 9% and 17% greater respectively than those for native BPTI. Cross sections for native BPTI were smaller than previous estimates from the crystal structure but in reasonable agreement with values calculated from the radius of gyration determined by x-ray scattering from solution BPTI. The increase in cross section for reduced BPTI over native BPTI is similar to that seen in molecular dynamics simulations. The results show that the disulphide bonds of BPTI contribute to the folding of the gas phase ions, but that even in the absence of disulphide bonds the protein ions maintain compact structures. Comparisons to relative areas calculated from hydrodynamic radii of BPTI in solution suggest that when disulphide linkages are removed, BPTI in the gas phase unfolds less than BPTI in solution.

Amino Acid Substitution↗

PubMed1998

Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions.

The acetic acid-induced unfolding of cytochrome c (cyt c) and apomyoglobin (aMb) are studied under equilibrium conditions by electrospray ionization (ESI) mass spectrometry (MS). The folding states of the proteins in solution are monitored by the charge state distributions that they produce during ESI. A tightly folded protein shows lower charge states than the same protein in an unfolded conformation. The ESI-MS data presented in this study show that during the denaturation of cyt c, only two distinct charge state distributions are observed. These can be attributed to the native and to the acid-unfolded conformation, respectively. In the transition region where the folded and the unfolded conformation are both present in solution, these two distributions are observed simultaneously, thus giving rise to a bimodal ESI mass spectrum. These data reflect a highly cooperative (two state) folding behavior. In contrast, the acid-induced unfolding of aMb is accompanied by gradual shifts in the maxima of the observed charge state distribution. This indicates a non-cooperative unfolding behavior involving multiple protein conformations. The observations made here suggest that ESI-MS might be a general method for assessing the cooperativity of protein unfolding transitions. This study also addresses the issue of 'secondary' solvent effects for ESI-MS studies on the acid-induced unfolding of proteins. These effects influence the ESI charge state distribution without being related to conformational changes of the protein in solution and could potentially complicate the interpretation of ESI mass spectra. Data obtained for bovine pancreatic trypsin inhibitor and ubiquitin indicate that secondary solvent effects influence the observed charge state distributions only to a very minor extent between pH 8.5 and 2.5.

Acetates↗

PubMed1998

Stability of a highly charged noncovalent complex in the gas phase: holomyoglobin.

Gas phase holomyoglobin (hMb) ions in charge states +7 to +21 were formed by electrospray ionization in combination with a continuous-flow mixing apparatus. Collision cross section measurements show that the highly charged ions are somewhat unfolded in comparison to low charge states but still retain a considerable degree of folding. A new collision model is presented which calculates the relative energies transferred to complexes in tandem mass spectrometry. Tandem mass spectrometry and ion trapping experiments both show that the energies required to dissociate heme from the highly charged heme-protein complexes in the gas phase are similar to those of low charge states, previously shown in literature ion cyclotron resonance experiments to be 0.7-1.0 eV. These energies are comparable to those of the heme binding energy in solution. The results suggest that even for the highly charged hMb ions which have unfolded somewhat, the heme-protein interactions remain relatively unperturbed.

Animals↗

PubMed1998

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, Biochemistry 1997, 36, 12,296-12,302) 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.

Cytochrome c Group↗

PubMed1998

Biosynthesis of domoic acid by the diatom Pseudo-nitzschia multiseries.

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with alpha-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate-mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate.

Acetates↗

PubMed1997

Acid-induced unfolding of cytochrome c at different methanol concentrations: electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure.

The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the alpha-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the acid-denatured states for the two methanol concentrations also show very similar mass spectra with a maximum at (cyt c + 17H+)17+. This indicates that the charge state distribution generated during ESI is not sensitive to the differences in the secondary structure of the denatured protein. The observed transition from low to high charge states is due to the breakdown of the tertiary structure in both cases. These findings suggest that ESI MS might be a general method to selectively monitor changes in the tertiary structure of proteins.

Animals↗

PubMed1997

Acid-induced denaturation of myoglobin studied by time-resolved electrospray ionization mass spectrometry.

The acid-induced denaturation of holo-myoglobin (hMb) following a pH-jump from 6.5 to 3.2 has been studied by electrospray ionization (ESI) mass spectrometry in combination with a continuous flow mixing technique (time-resolved ESI MS). Different protein conformations are detected by the different charge state distributions that they generate during ESI. The changes in intensity of the peaks in the mass spectrum as a function of time can be described by two exponential lifetimes of 0.38 +/- 0.06 s and 6.1 +/- 0.5 s, respectively. The acid-induced denaturation of hMb was also studied in stopped-flow experiments by monitoring changes in the Soret absorption. The lifetimes measured by this method are in good agreement with those obtained by time-resolved ESI MS. The shorter lifetime is associated with the formation of a transient intermediate which shows the mass of the intact heme-protein complex but leads to the formation of much higher charge states during ESI than native hMb at pH 6.5. This form of hMb has an absorption spectrum similar to that of the native protein, indicating a relatively unperturbed chromophore environment inside the heme binding pocket. The intermediate can thus be characterized as an unfolded form of hMb with essentially intact heme-protein interactions. The longer of the two lifetimes is associated with the formation of a product which has a blue-shifted absorption spectrum with a much lower maximum absorption coefficient than observed for native hMb. In the ESI mass spectrum, this product appears as the apoprotein with high charge states which indicates the disruption of the native heme-protein interactions and a considerable degree of unfolding compared to native apo-myoglobin. The mechanism of acid-induced denaturation of hMb, therefore, appears to follow the sequence (heme-protein)native --> (heme-protein)unfolded --> heme + (protein)unfolded.

Acetic Acid↗

PubMed1997

Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry.

A new method for studying the folding kinetics of proteins is described. The method combines a continuous flow mixing technique with an electrospray mass spectrometer. Different protein conformations in solution are detected by the different charge states they produce during electrospray ionization. Unfolded proteins generally have more accessible protonation sites and give higher charge states than native proteins. The method is applied to study the refolding of acid-denatured cytochrome c. Global data analysis is used to obtain the exponential lifetimes which are associated with the refolding process. The kinetics can be described by two lifetimes of 0.17 +/- 0.02 and 8.1 +/- 0.9 s which are in accordance with the results of stopped flow experiments previously described in the literature. These lifetimes are associated with roughly 90 and 10% of the total intensity changes in the mass spectrum, respectively, and most likely reflect fast and slow refolding subpopulations of cytochrome c in solution.

Acetic Acid↗

PubMed1997

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.

Animals↗

PubMed1995

Mass spectral analyses of microcystins from toxic cyanobacteria using on-line chromatographic and electrophoretic separations.

The application of capillary electrophoresis and of reversed-phase liquid chromatography coupled to electrospray mass spectrometry is presented for the analysis of microcystins isolated from toxic strains of Microcystis aeruginosa. The separation performance of these two techniques is compared in terms of both sensitivity and of resolution of closely related microcystins. Quantitation of microcystin-LR present at low micrograms/ml concentrations in cell extracts is demonstrated using both techniques. A marked advantage of capillary electrophoresis over liquid chromatography was its ability to resolve different desmethyl microcystin-LR analogues. Identification of these positional isomers was facilitated using capillary electrophoresis combined with tandem mass spectrometry (MS-MS). Rationalization of fragment ions observed in MS-MS spectra of microcystins was made possible through comparison with 15N labelled microcystins obtained from stable isotope feeding experiments. The potential of tandem mass spectrometry in providing selective detection of microcystins in cell extracts, and in structural characterization of novel microcystins, was also investigated.

Amino Acid Sequence↗

PubMed1995

Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudo-nitzschia multiseries.

Axenic cultures of Pseudo-nitzschia multiseries (formerly Pseudonitzschia pungens f. multiseries) produce less domoic acid (DA) than the original bacteria-containing cultures. Bacterial strains isolated from two nonaxenic P. multiseries clones were reintroduced individually into cultures of three axenic P. multiseries strains. The bacteria did not substantially affect division rates or cell yields. However, they did cause a 2- to 95-fold enhancement of DA production (per cell basis) relative to the axenic culture, depending on the P. multiseries and bacterial strain used. Bacteria isolated from a nontoxic Chaetoceros sp. culture also enhanced DA per cell (by 115-fold), showing that it is not necessary for the bacteria to be isolated from a toxic culture in order to enhance toxin production. There was no evidence of intracellular bacteria in disrupted P. multiseries cells obtained from axenic cultures. Our results demonstrate an important, but nonessential, role of extracellular bacterial in DA production. Characterization of the bacterial strains using morphology, substrate utilization, and restriction fragment length polymorphism (RFLP) analyses clearly showed that we had isolated different species of bacteria from the various nonaxenic cultures. We conclude that not one but several bacterial species enhance DA production by P. multiseries.

Cells, Cultured↗