Identification of Variants of the High Plains virus Infecting Wheat in Kansas.

The properties of two virus isolates (U04-82 and U04-83) obtained from two wheat (Triticum aestivum) plants expressing mosaic symptoms were investigated using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), time-of-flight mass spectrometry (TOFMS), and infection of wheat with resistance to Wheat streak mosaic virus (WSMV). The coat protein mass was estimated by SDS-PAGE as approximately 32 kDa for U04-82 and 30 kDa for U04-83. The amino acid sequence of the coat protein of U04-82 was 99.6 and 85.5% identical to two isolates, ABC58222 and TX96, respectively, of High Plains virus (HPV) described from Texas. U04-82 was transmitted by wheat curl mites and caused significant yield reductions in wheat resistant to WSMV. U04-83 was actually two distinct virus isolates whose capsid protein amino acid sequences were only 57 and 50% similar to that of TX96. Antiserum prepared to a synthetic peptide from the sequence of the U04-83 isolate recognized the two U04-83 isolates, but not the U04-82 isolate.

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.

Mass spectrometric study of the Escherichia coli repressor proteins, Ic1R and Gc1R, and their complexes with DNA.

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).

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.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

Characterization of plasma proteins adsorbed onto biomaterials. By MALDI-TOFMS.

The analysis of plasma proteins adsorbed onto a polyurethane (PU) biomaterial was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This article marks the first study on MALDI-TOFMS analysis of multiple proteins adsorbed from plasma, in vitro, onto the surface of a biomaterial to easily enable their characterization. Plasma standards from three different hosts were placed in contact with non-porous PU, a model biomaterial. Following the use of washing protocols developed in our laboratory, the biomaterial was analyzed, directly, with MALDI-TOFMS. Proteins with molecular weights (Mr) ranging from ca. 6.5 to 150 kDa were observed in the mass spectra and characterized upon comparison with proteins of known Mr. The proteins observed were tentatively identified as those known to adsorb onto PU, both in vitro and in vivo. In an attempt to model in vivo sorption, the PU biomaterial was exposed to freshly collected canine plasma, in vitro, for different lengths of time. Corresponding MALDI-TOFMS spectra displayed increasing protein signal for a number of different proteins with increasing times of exposure to plasma. This method provided qualitative and semi-quantitative analysis of the proteins adsorbed onto the biomaterial surface.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

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.

Characterization of a novel potyvirus isolated from maize in Israel.

A potyvirus (proposed name of Zea mosaic virus [ZeMV]) isolated from maize in Israel was analyzed by serology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of capsid proteins, symptomatology, and sequencing. Parts of the nuclear inclusion b, coat protein, and 3' regions were sequenced; the amino acid sequence of ZeMV capsid was determined by time-of-flight mass spectrometry (TOFMS). The results of these analyses were compared with those of similar analyses of the following potyviruses: Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus strain MDB (SCMV-MDB), Johnsongrass mosaic virus(JGMV), Sorghum mosaic virus (SrMV), and an isolate of MDMV from Israel. Indirect enzyme-linked immunosorbent assay using ZeMV antiserum detected only ZeMV, and reciprocal tests using MDMV, JGMV, or SrMV antisera failed to detect ZeMV. ZeMV cross-reacted weakly when SCMV-MDB antiserum was used. The mass of ZeMV capsid was determined to be 36,810 Da by SDS-PAGE and 34,216 Da by TOFMS. The ZeMV systemically infected johnsongrass (Sorghum halepense), but did not infect oat (Avena sativa), pearl millet (Pennisetum glaucum), barley (Hordeum vulgare), or rye (Secale cereale). Necrosis was caused in 19 sorghum lines by SrMV, in 15 by ZeMV, in 14 by MDMV, and in 5 by JGMV and SCMV-MDB. The nucleic acid and amino acid sequences of ZeMV clearly showed that it is not a strain of JGMV, MDMV, SCMV, or SrMV.

Biological and Molecular Variability Among High Plains virus Isolates.

The High Plains virus (HPV), vectored by the wheat curl mite (WCM) (Aceria tosichella), causes a severe disease of maize (Zea mays) in the U. S. High Plains. In the present study, five HPV isolates from five states were separated from co-infecting Wheat streak mosaic virus and their molecular and biological variability studied. Molecular studies involved time-of-flight mass spectrometry (TOFMS) to determine amino acid sequence variability of the 32-kDa nucleoprotein (32 np) of the isolates. Biological studies involved testing the ability of the five HPV isolates to infect a maize line previously shown to have resistance. Inoculations of the HPV isolates were conducted using vascular puncture inoculation (VPI) and viruliferous WCM. TOFMS analyses demonstrated an 18-amino acid sequence in the isolates at the N-terminus of the 32 np, the presence of amino acid sequence differences among the isolates, and variability among amino acid sequences of the 32 np of some isolates. Three of the five HPV isolates infected the resistant maize inbred, B73, using VPI, and two of the same three HPV isolates infected this line using WCM inoculation, albeit low numbers of plants were infected by each technique.

Characterization of a distinct Johnsongrass mosaic virus strain isolated from sorghum in Nigeria.

A virus isolated from sorghum in Nigeria has been partially characterized. It was tested by enzyme-linked immunosorbent assay (ELISA) using antisera to Maize dwarf mosaic virus, Johnsongrass mosaic virus (JGMV), Sugarcane mosaic virus strain-MDB, Sorghum mosaic virus, and Zea mosaic virus. A partial host range, symptom phenotypes for selected sorghum lines, and the mass of the coat protein (CP) subunit was analyzed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid (aa) sequence determined by time-of-flight mass spectrometry (TOFMS). The Nigerian isolate was positive in ELISA to only JGMV antiserum. It infected sorghum and smooth brome but not oat or johnsongrass. It caused necrosis in 12 of 13 tested sorghum lines, while the USA JGMV isolate caused necrosis in only one sorghum line. In SDS-PAGE, the mass of the Nigerian virus CP was 3,000 Da smaller than that of JGMV-MDO. Moreover, TOFMS analyses showed that, while residues 1-7 of the CP aa sequence were identical to those of JGMV (GenBank #A27631), and residues 57-293 were almost identical to residues 67-303 of JGMV, the intermediate region exhibited significant differences, including a 10 aa deletion. These data indicate that the virus should be considered a distinct isolate of JGMV (JGMV-N) and expands the known range of JGMV to Africa.

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.

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.

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.

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.

Peer Reviewed: Orthogonal-Injection TOFMS for Analyzing Biomolecules.

Orthogonal injection provides a high- efficiency interface for transferring ions from a continuous beam to a pulsed mode and makes it easier to obtain high resolution.

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)

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.