Complex hprt deletion events are recovered after exposure of human lymphoblastoid cells to high-LET carbon and neon ion beams.
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Hypoxanthine phosphoribosyltransferase gene (hprt) mutations were induced in human TK-6 lymphoblastoid cells by irradiation at a linear energy transfer (LET) of 250 or 310 keV/micron for carbon and neon ions, respectively. At such a high level of LET, ions will lose most of their total energy and stop shortly after passing through the cell. The hprt mutations were analyzed by multiplex PCR, long-PCR and DNA sequencing of both genomic and cDNA. Over half of the C ion-induced hprt mutations (10 of 19) were point mutations, in contrast to 15% of the mutations induced by Ne ions (three of 20). The remaining 47 and 85% of the C and Ne ion-induced mutants, respectively, are deletion events. The latter events include three complex losses of multiple non-contiguous exon regions in both ion irradiation collections. We note that mutations involving the exon 6 region are frequent in the Ne ion collection: all three of the complex events retained the exon 6 region with flanking deletion of sequence and three other mutants involved deletion of this region. It may be concluded that these high-LET C and Ne ion irradiations produce different mutational spectra. (+info)
Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca(2+)-induced conformational changes in the regulatory domain of human cardiac troponin C.
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Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis. (+info)
Formation and characterization of iron-oligonucleotide complexes with matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry.
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Iron-containing oligonucleotide negative ions can be generated by matrix-assisted laser desorption/ionization from a stainless steel target disk (by either defocusing the laser beam or by mixing iron salts such as FeCl3 with the matrix compound during the sample preparation). High resolution mass measurements reveal the presence of both Fe2+ (as M + Fe - 3H)- and Fe3+ (as M + Fe - 4H)- in the metal-oligonucleotide ions. The presence of Fe3+ is unexpected, and must involve replacement of protons from the nucleic bases or ribose groups as well as the phosphate groups of the oligonucleotides. Inspection of a range of small oligonucleotides and mononucleotides reveals that the presence of both Fe2+ and Fe3+ in the iron-biomolecule complexes is dependent on the number of acidic hydrogens that can be replaced in the oligonucleotide or nucleotide. Collisional dissociation of several metal-tetranucleotide ions revealed that the presence of the iron ion alters the fragmentation observed. The iron atom was observed to be present in all of the fragment ions, and, whenever possible, seemed to enhance the abundance of fragment ions containing both iron and a guanine nucleic base. These results suggest that iron may serve as a useful probe for characterizing phosphorylated biomolecules. (+info)
Laser desorption in transmission geometry inside a Fourier-transform ion cyclotron resonance mass spectrometer.
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We report here the first application of laser desorption (LD) in transmission geometry (backside irradiation of the sample through a transparent support) inside a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). A probe-mounted fiber optic assembly was used to simplify the implementation of this LD technique. This setup requires little or no instrument modifications, has minimum maintenance requirements, and is relatively inexpensive to build. The performance of the probe was tested by determining the molecular weight of a commercial polystyrene standard from its matrix-assisted laser desorption/ionization (MALDI) spectrum. The measured average molecular weight is comparable to that obtained for the same sample by MALDI in the conventional top-illumination arrangement (reflection geometry) and by the manufacturer of the sample by gel permeation chromatography. The average velocities measured for ions evaporated by transmission mode LD of several neat samples are about half the velocity of those obtained by using the reflection geometry. Therefore, transmission mode irradiation of the sample holds promise to desorb ions that are easier to trap in an ICR cell. An oscillating capillary nebulizer was adapted for the deposition of analytes to improve sampling reproducibility. (+info)
Determination of 3,3'-dichlorobenzidine and its degradation products in environmental samples with a small low-field Fourier transform ion cyclotron resonance mass spectrometer.
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3,3'-Dichlorobenzidine (DCB) and its degradation products, 3-chlorobenzidine (MCB) and benzidine, are of environmental concern because of their carcinogenic nature. The suitability of a small Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer for the analysis of these environmental contaminants in different types of matrices was explored. All the measurements were carried out by depositing the sample solution directly on a disk that was introduced into the mass spectrometer. This approach is very fast and simple because it requires no prior chromatographic separation or derivatization. Calibration curves determined by collecting 70-eV electron ionization mass spectra of neat samples yielded lower limits of detection of 29 and 17 pg (total amount on the solids probe) for DCB and benzidine, respectively (based on a signal to noise ratio of > or = 2:1), while chemical ionization with ammonia resulted in lower limits of detection of 21 pg for DCB and 9 pg for benzidine (total amount on the solids probe). FT-ICR analysis of sediments collected from Lake Macatawa (Holland, MI) verified the presence of DCB in this complex, environmentally significant sample matrix. Laboratory experiments designed to probe biodegradation and photodegradation pathways showed that DCB undergoes sequential dehalogenation to yield MCB and then benzidine under exposure to microorganisms and under simulated tropospheric solar radiation. The ability of the FT-ICR to determine elemental compositions of compounds introduced as described above was demonstrated for one of the degradation products. (+info)
Initial implementation of an electrodynamic ion funnel with Fourier transform ion cyclotron resonance mass spectrometry.
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Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has become a widely used method to study biopolymers. The method, in combination with an electrospray ionization (ESI) source has demonstrated the highest resolution and accuracy yet achieved for characterization of biomolecules and their noncovalent complexes. The most common design for the ESI interface includes a heated capillary inlet followed by a skimmer having a small orifice to limit gas conductance between a higher pressure (1 to 5 torr) source region and the lower pressure ion guide. The ion losses in the capillary-skimmer interface are large (estimated to be more than 90%) and thus reduce achievable sensitivity. In this work, we report on the initial implementation of a newly developed electrodynamic ion funnel in a 3.5 tesla ESI-FTICR mass spectrometer. The initial results show dramatically improved ion transmission as compared to the conventional capillary-skimmer arrangement. An estimated detection limit of 30 zeptomoles (approximately 18,000 molecules) has been achieved for the analysis of the proteins with molecular weights ranging from 8 to 20 kDa. (+info)
Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor beta and derivatives.
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The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor beta (rhM-CSF beta) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (approximately 25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods. (+info)
Electrospray ionization Fourier transform ion cyclotron resonance analysis of large polymerase chain reaction products.
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In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR. (+info)