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K G Standing

Publications and source records attributed to K G Standing.

At least 37 records · Page 2Linked to original sources
PubMed1998

Electrospray mass spectrometry studies of non-heme iron-containing proteins.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

Mass Spectrometry↗
PubMed1998

Sensitive high-resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time-of-flight mass spectrometry.

Off-line and on-line capillary zone electrophoresis-electrospray ionization time-of-flight mass spectrometry (CZE-ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI-TOF-MS at the pmol level. Sheathless CZE-ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE-ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/N values of 250-400 on raw data (TIE mode) and of 30-760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 (m/delta(m)(FVHM)) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35000-87000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800-5500 and S/N values of 110-225 on raw data. The gold-coated sheathless CE-ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.

Acetic Acid↗
PubMed1998

Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry.

A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms. An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form. The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants. The measured constants agree with those determined by conventional means.

Chemical Phenomena↗
PubMed1998

Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.

Bacterial Proteins↗
PubMed1997

Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.

Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

Databases, Factual↗
PubMed1997

Use of a non-porous polyurethane membrane as a sample support for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins and peptides was performed on samples deposited onto non-porous ether-type polyurethane (PU) membranes. Spectra obtained using PU membranes showed that mass resolution and accuracy were equivalent to values observed using a metal target, and superior to those obtained using poly(vinylidene difluoride) (PVDF) membranes. A small apparent increase in the mass of proteins and also loss of resolution were observed at very high laser irradiance due to charging, but were not observed under normal conditions. Analysis of NaCl-doped standards demonstrated that PU membranes yielded better results than a metallic target for salt-containing solutions. Relatively strong hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step. This step allowed for the removal of salts and buffer components and thus provided an increase in resolution and mass accuracy. Digestion of citrate synthase (a protein of molecular weight 47,886) with trypsin was performed directly on the surface of the membrane for variable periods of time, and characteristic peptide fragments were observed by MALDI-TOFMS. Delayed extraction was used to increase the resolution and to permit more accurate mass assignments for those fragments. The use of PU membranes for MALDI-TOFMS analysis of proteins with higher molecular weights is also demonstrated.

Citrate (si)-Synthase↗
PubMed1996

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Circular Dichroism↗
PubMed1996

Preparation and properties of pure, full-length IclR protein of Escherichia coli. Use of time-of-flight mass spectrometry to investigate the problems encountered.

IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue). Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene.

Amino Acid Sequence↗
PubMed1994

Reflecting time-of-flight mass spectrometer with an electrospray ion source and orthogonal extraction.

An electrospray source has been coupled to a reflecting time-of-flight mass spectrometer. The ions enter as a continuous beam in a direction perpendicular to the spectrometer axis and are formed into short bursts of ions with velocities parallel to the axis by electrical pulses applied to injection electrodes. The instrument may be operated either in the linear mode, with ions detected behind the electrostatic mirror, or in the reflecting mode, with ions detected after reflection. In the latter case the time resolution is < or = 8 ns for m/z approximately 600, enabling observation of individual isotopic peaks for masses up to about 4000 u. The sensitivity is adequate to enable measurement of mass spectra for 10 fmol of cytochrome c (approximately 12,000 u). The spectrometer does not limit the range of m/z values, and ions have been observed up to m/z approximately 6000. With proper adjustment of pH and other conditions in the source, the ionization is very soft, enabling injection of weakly bound complexes; these have been accelerated and measured in the spectrometer without observable fragmentation.

Cytochrome c Group↗
PubMed1994

Investigation of protein-protein noncovalent interactions in soybean agglutinin by electrospray ionization time-of-flight mass spectrometry.

Noncovalent interactions in soybean agglutinin (SBA) were studied on an electrospray ionization (ESI) time-of-flight mass spectrometer constructed recently at the University of Manitoba. The high m/z range and high sensitivity of the instrument together with mild ESI interface conditions turned out to be ideal for detecting this noncovalently bonded tetrameric protein (MW approximately 116,000 Da) in low charge states (z = 23 to 27). By altering the acetonitrile content of the SBA solutions it was shown that the observed SBA tetramers are due to structurally specific noncovalent associations in solution. Octamers and dodecamers (MW approximately 350,000 Da) were also detected. Information on the quaternary structure of the tetramers was obtained by analyzing the fragment-ion spectrum resulting from the collision-induced dissociation of the tetramer ions.

Lectins↗
PubMed1993

Laser debonding of ceramic orthodontic brackets.

Laser light energy has been shown in other studies to degrade resins by thermal softening, thermal ablation, or photoablation. If this technology could be successfully applied to bracket debonding, fracturing of both bracket and enamel during debonding might be eliminated. Both polycrystalline alumina and single crystal alumina (sapphire) ceramic orthodontic brackets were bonded to the labial surfaces of lower deciduous bovine incisor teeth with the acid-etch technique as currently practiced in dentistry. Under an externally applied stress of either zero or 0.8 MPa, the brackets were debonded by irradiating the labial surfaces of the brackets with laser light at wavelengths of 248 nm, 308 nm, and 1060 nm, and at light power densities of between about 3 and 33 W/cm2. Debonding times were measured, and the surfaces created by debonding were examined with both light and scanning electron microscopy to determine the extent of bracket and enamel damage. The results showed that under the conditions of this study, no enamel or bracket damage was present in any sample. The polycrystalline brackets debonding times were about 3 seconds, 5 seconds, and 24 seconds for 248 nm, 308 nm, and 1060 nm of radiation, respectively. The debonding of polycrystalline brackets is caused by thermal softening of the bonding resin resulting from heating of the bracket. The hot bracket then slides off the tooth. All sapphire brackets debonded in less than 1 second. At sufficiently high power levels, debonding of sapphire brackets is caused by either thermal ablation or photoablation resulting from direct interaction of the light beam with the resin.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1992

Secondary-ion and electron production from surfaces bombarded by large polyatomic ions.

Heavy molecular ions with energies in the range 10-20 keV and masses from 276 u to 132,000 u, produced by matrix-assisted laser desorption, were used as primary projectiles to produce secondary-ion spectra from a variety of surfaces in a tandem time-of-flight mass spectrometer. In the negative mode the ratio of electron emission to secondary-ion emission was found to decrease rapidly with increasing projectile mass. Ion emission was found to dominate for primary ions larger than approximately 10,000 u. Positive or negative molecular ions and cations were observed from several organic targets of masses up to 1140 u (gramicidin S) for incident projectiles up to mass 132,000 u, i.e., for projectile speeds down to approximately 7000 m/s. Other ions characteristic of the target were also observed for these projectiles. Thus, large polyatomic ions can cause secondary-ion desorption even at very low velocity. The background ions of both polarities are similar to those found in keV particle bombardment by monatomic projectiles. The same ions are observed for all the projectiles; most can be identified with hydrocarbon background. The relative intensities of the background positive ions are largely independent of projectile, and for both polarities the ratio of the ions characterizing the target to those forming the background is approximately constant for all the projectiles. These results strongly suggest that the background ions come from the usual layer of organic impurities attached to the target surface. No direct evidence for surface-induced dissociation was observed in this mass and energy range.

Mass Spectrometry↗
PubMed1992

Kinetic energy measurements of molecular ions ejected into an electric field by matrix-assisted laser desorption.

Measurements of kinetic energy distributions of molecular ions ejected into an extraction field by matrix-assisted laser desorption are reported. The measurements were made in a time-of-flight mass spectrometer with an electrostatic mirror by measuring the reflected signal as a function of the difference between the accelerating voltage and the voltage applied to the mirror. The molecular ions were found to have less kinetic energy than the extraction field alone would normally provide, i.e., we observed an energy deficit. Under conditions typical for a matrix-assisted laser desorption experiment, the deficit is about 24 eV for molecular ions of insulin. The size of the deficit increases with the intensity of the molecular ion signal, and the molecular weight of the protein; it is also larger for negative molecular ions than for positive molecular ions.

Animals↗
PubMed1991

Properties of matrix-assisted laser desorption. Measurements with a time-to-digital converter.

Some properties of matrix-assisted laser desorption have been studied using single-ion-counting methods and a time-to-digital converter. The methods allow examination of the process for irradiances near the reported threshold for observation with a transient recorder. All measurements were made using bovine insulin as a test compound. We present direct evidence that an irradiance threshold near 10(6) W cm-2 exists for ion production, and that the process is a collective effect, either involving a large number of molecular ions (approximately 10(4) in a successful event or none at all. Above the threshold, the yield is found to scale with a high power (4th to 6th) of the irradiance. Measurements of initial velocity distributions indicate an axial velocity spread corresponding to approximately 50 eV and a radial velocity spread corresponding to approximately 2.4 eV. Thus the ejection or extraction mechanism appears to be strongly asymmetric.

Animals↗
PubMed1989

Measurement of unimolecular decay in peptides of masses greater than 1200 units by a reflecting time-of-flight mass spectrometer.

Daughter ions from decomposition of [M + H]+ parent ions have been observed in a time-of-flight mass spectrometer fitted with an ion mirror. Unit mass resolution is obtained for parent ions of masses up to several thousand u when the mirror voltage is set at a value determined by the usual velocity-focusing criterion for parent ions. Under this condition the daughter-ion resolution in the low-mass range suffers. Considerable improvement is obtained when the daughter-ion spectrum is examined in several segments, with the mirror voltage optimized for each segment. The daughter-ion spectrum from decay of metastable [M + H]+ parent ions in Substance P is measured using this technique. Reasons are suggested for differences between the spectrum obtained and a spectrum for the same compound recently obtained using a tandem double-focusing instrument.

Endorphins↗
PubMed1987

Redox reactions occurring during secondary ion mass spectrometry of some heteroanthracycline antitumor antibiotic precursors.

Positive and negative secondary ion mass spectra of the title compounds were produced by bombardment of solid deposits by 13 and 23 KeV cesium ions, respectively, and analyzed with a time-of-flight mass spectrometer. In addition to familiar protonation and alkali (mainly sodium) ion addition processes observed in the positive ion spectra and proton abstractions in the negative ion spectra (which enable determination of molecular weight) there is evidence from both spectral modes for oxidation-reduction processes in which species such as [M - H]+, [M - H + Na]+. and [M + 1]- are formed. It is argued that these may be formed rapidly from pre-associated molecules during bombardment/ionization rather than as a result of radiation damage. Sodium ion addition is apparently facilitated by the presence of electron pair donor groups, especially if chelation is possible.

Antibiotics, Antineoplastic↗