Fragmentation Mass Spectra Research Articles

Thermodynamic interpretation of the uniformity of the phase space probability measure

Uniformity of the probability measure of phase space is considered in the framework of classical equilibrium thermodynamics. For the canonical and the grand canonical ensembles, relations are given between the phase space uniformities and thermodynamic potentials, their fluctuations and correlations. For the binary system in the vicinity of the critical point the uniformity is interpreted in terms of temperature dependent rates of phases of well defined uniformities. Examples of a liquid-gas system and the mass spectrum of nuclear fragments are presented.

Structural Characterization of the Cysteine-rich Domain of TFIIH p44 Subunit

In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.

Identification of non-covalent structure in apocytochrome c by hydrogen exchange and mass spectrometry.

Apocytochrome c, the in vivo precursor to active cytochrome c, was analyzed by amide hydrogen exchange and mass spectrometry to search for fixed, non-covalent structure. The protein was incubated in H(2)O at pH 3.3 or 6.7 for various times, then exposed to D(2)O to initiate isotope labeling of unfolded regions. Following acid quenching of hydrogen exchange, the labeled apocytochrome c was digested with pepsin into fragments that were analyzed by directly coupled high-performance liquid chromatography/electrospray ionization mass spectrometry. The intermolecular distribution of deuterium and the deuterium levels in structurally distinctive populations were determined from the mass spectra of the peptic fragments. Spectra of peptic fragments derived from apocytochrome c incubated at pH 3.3 had single envelopes of isotope peaks with masses indicating that all of the amide hydrogens had been replaced with deuterium. These results showed that apocytochrome c at pH 3.3 offered little resistance to hydrogen exchange, indicating that it was unfolded with little fixed structure. However, mass spectra of peptic fragments including residues 81-94 of apocytochrome c incubated at pH 6.7 had two envelopes of isotope peaks, indicating that one population was unfolded and the other population was highly structured in this region. Mass spectra of peptic fragments including residues N-terminal to residue 81 indicated that this region of the protein remained unfolded with little fixed structure at pH 6.7.

Electrospray–mass spectrometry characterization and measurement of far-UV-induced thymine photoproducts

Far-UV-induced formation of dimeric pyrimidine photoproducts within DNA is a major cause of the carcinogenic effects of solar light. The chemical structure of this class of lesion has been mostly determined by studies on model compounds. The present work is aimed at providing mass spectrometry data on the thymine–thymine photoproducts, including the diastereoisomers of the cyclobutane dimer, the (6–4) adduct, the related Dewar valence isomer and the spore photoproduct. Fragmentation mass spectra of the modified bases, nucleosides, dinucleoside monophosphates and dinucleotides were recorded following electrospray ionization with either triple-quadrupolar or ion-trap detection. The results showed differences in fragmentation pattern between the different types of photoproducts. In addition, a drastic effect of the diastereoisometry was observed for the cyclobutane dimers. A sensitive detection technique has been developed for the analysis of dinucleoside monophosphate photoproducts by high-performance liquid chromatography associated with mass spectrometry in the negative mode with multiple reaction-monitoring detection.

Duality Monomer–Dimer of the Pheromone-Binding Protein from Bombyx mori

The analysis of a recombinant pheromone-binding protein from the silkworm moth, Bombyx mori, by native gel electrophoresis with Coomassie staining showed one single band with a molecular mass consistent with a monomer. A slow migrating band, detected in the recombinant and native samples by a polyclonal antibody, was indistinguishable from the monomer in the mass spectrum fragmentation pattern and chromatographic behavior. Flow injection analyses of the protein by mass spectrometry in the negative mode showed fragments of a dimer. The dimeric form was also supported by estimation of the molecular mass by gel filtration at basic pH. A cross-linked dimer coeluted with the noncovalent dimer on a gel filtration column. The molecular mass of the protein changed in a pH-dependent way with a dramatic transition from dimer to monomer between pH 6 and 4.5. A low pH induced not only dissociation of the dimer, but also a conformational change in the protein. In marked contrast to denaturation with guanidinium chloride, the emission maxima of tryptophan was not significantly changed at low pH. BmPBP is thus a dimer at slightly acid, neutral, and basic pH, which dissociates and then undergoes conformational change at low pH.

Post-source decay fragmentation analyses of linkage isomers of Lewis-type oligosaccharides in curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: combined in-source decay/post-source decay experiments and relative ion abundance analysis

Linkage isomers of Lewis(X) trisaccharide (Le(X)) and Lewis(a) trisaccharide (Le(a)) were distinguished by the post-source decay (PSD) fragment spectra obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without permethylation. Both Y- and Z-type fragmentations were observed at the C-3 position of N-acetylhexosamine. beta-Elimination at C-3 of the reducing-end N-acetylglucosamine in Le(X) formed a double bond, which conjugated to an N-acetyl group, making the chemical species stable. In contrast, the double bond formed in the reducing end glucose of 3-fucosyllactose was unstable owing to the lack of a conjugated system. Therefore, beta-elimination of N-acetylglucosamine occurred predominantly rather than that of hexose in MALDI-PSD fragmentation. The measurements of the PSD fragment mass spectra using pseudo precursor ions originating from in-source decay were useful for the analyses of the fragmentation mechanisms and for the assignments of the chemical species of the fragment ions. The combined in-source decay/post-source decay experiments revealed the formation of a double bond between C-2 and C-3 in N-acetylglucosamine of Le(X). Abundance analysis of the PSD ions indicated that the 1-3 glycosyl linkage cleaves more easily than does the 1-4 linkage in MALDI-PSD fragmentation. Ion abundance analyses were useful in estimating the degree of Y- and Z-type fragmentation at the C-3 position of hexose and N-acetylhexosamine. The analysis of the relative ion abundances was a powerful tool for the assignments of the chemical species of the PSD ions.

Analysis of toxic norditerpenoid alkaloids in Delphinium species by electrospray, atmospheric pressure chemical ionization, and sequential tandem mass spectrometry.

A rapid electrospray mass spectrometry method was developed for screening larkspur (Delphinium spp.) plant material for toxic norditerpenoid alkaloids. The method was calibrated using two standard alkaloids, methyllycaconitine (1) and deltaline (2), with a recovery of 92% from spiked samples and relative standard deviations of 6.0% and 8.1% for the two alkaloids, respectively. Thirty-three samples of plains larkspur, Delphinium geyeri, were analyzed. Methyllycaconitine (1) concentration was 0.27% +/- 0.08% during a 1-month period in 1997 establishing the relative risk of poisoning from the plant to be low. The method was also applied to the trace analysis (<1 ppm) of 1 in serum samples from sheep dosed different levels of the alkaloid. Electrospray ionization combined with sequential tandem mass spectrometry and HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry were used to detect and tentatively identify three new norditerpenoid alkaloids from Delphinium nuttallianum [bearline (6), 14-acetylbearline (7), 16-deacetylgeyerline (8)]. The tentative structure of the new alkaloids was predicted from the tandem mass spectra fragmentation patterns and assigning the substitution pattern for methoxy and acetyl groups at the C-14 and C-16 carbons.

Hydrogen exchange demonstrates three domains in aldolase unfold sequentially

Rabbit muscle aldolase is a homotetramer in which the subunits have a classical α/β-barrel structure and Mr 39,212 Da. We have previously reported that aldolase incubated in 3 M urea has three unfolding domains distinguished by their different unfolding rates. The unfolding rates of these domains were determined from isotope patterns in the mass spectra of peptic fragments derived from aldolase incubated in 3 M urea and pulse labeled in 2H2O. The present study extends this investigation to more thoroughly characterize the structures of these unfolding intermediates. Mass spectra of intact monomers had four envelopes of isotope peaks corresponding to four structural forms of aldolase. Analysis of the present results suggests that these structural forms consist of native aldolase and three forms that have one to three domains unfolded. The molecular masses of these four structural forms indicate that there are 107 residues in each of the three unfolding domains of aldolase. Present results also show that aldolase remains a tetramer in 4 M urea, even though hydrogen exchange and circular dichroism indicate that it has lost most of its secondary and tertiary structure. The abundances of unfolded domains, which were determined from mass spectra of either intact aldolase or its peptic fragments, were used to determine the abundances of specific, partially unfolded forms of aldolase. Kinetic modeling of the abundances of these structures suggests that these structures are formed sequentially as aldolase unfolds in urea.

Reaction of metformin with dicarbonyl compounds. Possible implication in the inhibition of advanced glycation end product formation.

Dicarbonyl compounds such as methylglyoxal and glyoxal are extremely reactive glycating agents involved in the formation of advanced glycation end products (AGEs), which in turn are associated with diabetic vascular complications. Guanidino compounds such as aminoguanidine appear to inhibit AGE formation by reacting with alpha-dicarbonyl compounds. The aim of this work was to study whether the antihyperglycemic agent metformin (a guanidine-like compound) might react with reactive alpha-dicarbonyls. Metformin was incubated at pH 7.4 and 37 degrees in the presence of either methylglyoxal or glyoxal and reaction products analysed by HPLC coupled to mass tandem spectrometry. AGE formation on albumin by methylglyoxal and glyoxal in the presence or absence of metformin was also studied by measuring the fluorescence at 370/440 nm after albumin-AGE isolation by ultrafiltration. As a standard for mass spectra analysis, a metformin-methylglyoxal adduct was chemically synthesised and characterised as a triazepinone (2-amino-4-(dimethyl-amino)-7-methyl-5,7-dihydro-6H-[1,3,5]triazepin+ ++-6-one). The results obtained showed that metformin strongly reacted with methylglyoxal and glyoxal, forming original guanidine-dicarbonyl adducts. Reaction kinetic studies as well as mass fragmentation spectra of the reaction products were compatible with the presence of triazepinone derivatives. In the presence of metformin, AGE-related fluorescence after albumin incubation with either glyoxal or methylglyoxal was decreased by 37% and 45%, respectively. These results suggest that besides its known antihyperglycemic effect, metformin could also decrease AGE formation by reacting with alpha-dicarbonyl compounds. This is relevant to a potential clinical use of metformin in the prevention of diabetic complications by inhibition of carbonyl stress.

Casein kinase II is responsible for phosphorylation of NF-L at Ser-473

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3β nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.

Surface-Induced Dissociation of Small Carbon Cluster Negative Ions (Cn-, n = 5−12): Correlation between the Dissociation Patterns and Stability of Fragment Ion−Neutral Pairs

We report detailed study of surface-induced dissociation (SID) of small carbon cluster negative ions (Cn-, n = 5−12) in collision with Si substrates. Fragment mass spectra have been observed in the collision energy range of 0−10 eV/atom. Neutral C3 loss occurred at collision energies lower than 2 eV/ atom. In the medium energy range (2−6 eV/atom), a marked even−odd difference in the dissociation has been observed: even fragment ions were predominantly generated from incident odd clusters, whereas both even and odd fragment ions from incident even clusters. These results are qualitatively in good agreement with the formation energies of fragment ion−neutral pairs calculated using the experimentally determined binding energies of ions and neutrals. At elevated collision energies (6−10 eV /atom), further fragmentation and electron detachment from fragment ions occurred and smaller cluster ions with even n were preferentially generated.

Development of a direct current gas sampling glow discharge ionization source for the time-of-flight mass spectrometer

A direct current, reduced-pressure, gas sampling glow discharge (GSGD) ionization source has been developed and interfaced to an orthogonally extracted time-of-flight mass spectrometer for the purpose of generating both atomic and molecular fragmentation mass spectra. The discharge is contained within the first vacuum stage of the differentially pumped interface of the mass spectrometer. The source is mechanically and logistically simple to construct, operate, and maintain. Switching between the atomic and molecular modes of operation is achieved by altering the discharge gas composition, the operating pressure, and the current. Helium was used to generate atomic mass spectra, whereas molecular spectra were produced by use of argon. Gas flow rates were less than 1 l min –1 for each mode of ionization. This report focuses primarily upon the atomic (elemental) analytical capabilities of the GSGD interface. Atomic detection limits are in the range of 20-90 pg s –1 (as the halogen) for analytes introduced into the system with an exponential dilution device, and with boxcar averagers employed for data collection. Precision is better than 0.4% relative standard deviation (RSD) for measurement of the 79 Br + / 81 Br + isotope ratio (presented to the source as bromoform vapor) over a period of 2.5 h. A variety of chlorinated hydrocarbons were introduced into the discharge via a flow cell, and it was possible to differentiate (i.e., speciate) the compounds based upon their 35 Cl + / 12 C + elemental ratios with a correlation coefficient (R) of 0.996.

Molecular dynamics studies of inelastic scattering and fragmentation in collisions of with rare-gas atoms

Collisions of with neon and helium atoms were studied using molecular dynamics with empirical two- and three-body forces. Inelastic scattering including the capture of the rare-gas atom and fragmentation were analysed at collision energies in the centre-of-mass system between 1 and 200 eV with sufficient statistics for the microscopic calculation of differential cross sections on an absolute scale. The angular distributions are characterized by enlarged average deflections of the rare-gas atom due to the surface corrugation at low collision energies and shooting through the at high collision energies . The kinetic-energy spectra of the scattered rare-gas atoms indicate the extreme inelasticity of in collisions. Good agreement between calculated and measured kinetic-energy spectra from collisions was found. In particular, the position of the inelastic peak at the low-energy end of the spectrum was reproduced perfectly. Cross sections for the formation of compounds were calculated in order to provide a measure for the fine tuning of potential parameters. The calculated mass spectra of the primary fragments at 200 eV collision energy exhibit a bimodal distribution with a few fission-like events in collisions. Double differential cross sections for the kinetic energy of cluster fragments formed as a direct result of the impact demonstrate the non-thermal character of this direct fragmentation of at high collision energies.

Fragmentation spectroscopy of photoionized Fe(CO) 5: a molecular model for a heterogeneous cluster

Protoionization experiments have been performed to investigate the fragmentation process of iron carbonyls (Fe(CO) 5) after absorbing a single photon of known energy. The used energy area was from 9.5 to 25 eV. A threshold photoelectron photoion coincidence (TEPICO) technique was used to obtain time-of-flight (TOF) mass spectra of different ionic fragments. It has been shown that the dynamics of the fragmentation depends strongly on the energy of the photon. Particularly noticeable is the small kinetic energy release of the larger fragments, which indicates a deposition of a large amount of energy into the vibrational modes of the fragment ions.

Electron impact ionization of spatially oriented CH 3Cl and CCl 3H

The influence of molecular orientation on 200 eV electron impact ionization has been probed with oriented target molecules in crossed particle beams. Ionization of oriented CH 3Cl to form the molecular ion, CH 3Cl +, is favoured for electron impact on the positive end of the dipole, the CH 3 end. Formation of the CH 3 + fragmentation ion shows no preference for either end of the dipole which has been interpreted as favouring broadside collisions with the molecule. The ionization cross-section and the fragmentation pattern or mass spectrum for CH 3Cl therefore exhibit an orientation dependence. Ionization of oriented CCl 3H to form the fragmentation product CCl 2H + is favoured for electron impact at the positive end, the H end, of the molecule. An electrostatic or Coulomb model for electron impact ionization which reproduces the main experimental features is introduced.

Thermospray liquid chromatography/mass spectrometry study of diastereomeric isoindole derivatives of amino acids and amino acid amides

A thermospray liquid chromatography/mass spectrometry (TSP-LC/MS) method is described for determination of the enantiomeric excess of α-amino acids and α-amino acid amides as their o- phthalaldehyde/N- acetyl- l-cysteine (OPA/NAC) derivatives. The source temperature is an important factor in optimizing the sensitivity of the TSP-LC/MS analysis, whereas the repeller voltage is of minor importance. On-column mass spectra were acquired for the OPA/NAC derivatives of several α-amino acids and α-amino acid amides. For the main fragment ions, mass spectra fragmentation pathways are proposed. The applicability of the method is demonstrated by means of the enantiomeric excess determination of valine in a sample from an enzymatic hydrolysis experiment. Using single ion monitoring, the detection limit of d-valine in the presence of excess l-valine is 10 pmol. The present TSP-LC/MS method is useful for validating the results obtained from LC/UV or LC/fluorescence methods for the enantiomeric excess determination of α-amino acids and α-amino acid amides.

Characterization of Braun's lipoprotein and determination of its attachment sites to peptidoglycan by 252Cf-PD and MALDI time-of-flight mass spectrometry

A strategy for the characterization of bacterial lipoprotein-in this case Braun's lipoprotein (an outer membrane 7-ku lipoprotein) isolated from Escherichia coli-is described by time-of-flight mass spectrometric (TOF/MS) techniques [(252)Cf plasma desorption (PD) TOF/MS and matrix-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkage of lipid at the N-terminal cysteine (posttranslationally modified to a S-[2,3-bis(acyloxy)-propyl]-N-acylcysteine) and, therefore, strict insolubility in aqueous solution constitute common features for this class of proteins. Relative molecular mass determination of the major molecular species of Braun's lipoprotein was obtained by selection of an appropriate mixture of organic solvents compatible with matrix/support materials useful for the mass spectrometric techniques applied. Minor components of this lipoprotein that differ only in the fatty acid composition of the lipid anchor were detected by PD TOF/MS after enzymatic release of the extremely hydrophobic N-terminal amino acid followed by selective extraction with chloroform. Part of the primary sequence of this lipoprotein was confirmed based on peptide fragment ions observed in the positive ion PD mass spectra of cyanogen bromide-generated peptide fragments that had been isolated previously by reverse phase high-performance liquid chromatography (HPLC). Peptidoglycan fragments that represent the attachment sites of lipoprotein to peptidoglycan were enzymatically released, separated by reverse phase HPLC, and finally characterized by time-of-flight mass spectrometric techniques ((252)Cf-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques differed only in the better sensitivity obtained with MALDI TOF/MS, which consumed a factor of 100 to 1000 less material than with PD TOF/MS.

Pyrolysis gas chromatography-mass spectrometry of natural resins used for artistic objects

Eight resins used as protective layers for paintings, small sculptures, jewelries and artistic furniture, namely, Manila copal, colophony, Venice turpentine, elemi, shellac, dammar, sandarac and mastic were subjected to pyrolysis gas chromatography-mass spectrometry (PY-GC-MS). Significant mass spectral ions were monitored in each pyrogram. A table for identification of such resins based on the most significant ions in the mass spectra of the pyrolysis fragments is proposed. The results of PY-GC-MS analysis of a sample from the earth globe Giovanni Enriques, attributed to the Venetian geographer Vincenzo Coronelli (1650–1718) (Museo della Specola, Department of Astronomy, University of Bologna), are reported as an example of practical application of the method to a real artistic sample.

Photo-assisted direct writing of conducting aluminum

We have succeeded in selective area deposition of aluminum films by ultraviolet photolytic decomposition of the organometallic complex trimethylamine alane, TMAA-AlH 3(N(CH 3) 3) 2. Both ultraviolet and visible light successfully photolysed the alane adduct and resulted in selective area deposition. The light sources exhibited different laser fragmentation mass spectra, suggesting somewhat different decomposition pathways.

Poincaré covariant particle dynamics. I. Intranuclear cascade model.

To analyze the high energy heavy ion reactions performed at Lawrence Berkeley BEVALAC, Brookhaven AGS, and CERN SPS we present a dynamical model that is entirely Poincar\'e covariant. The description of strong interactions by Lorentz-scalar quasipotentials makes possible a relativistic extension of the cascade concept without losing its inherent simplicity. No field degrees of freedom appear explicitly, neither in elastic nucleon scattering nor in particle creation processes. Although various formalisms describe directly interacting relativistic particles with identical solutions for the two-particle case, they all suffer from different problems in many-particle systems. The basis of our approach is a Hamiltonian formulation for N pointlike nucleons, moving unconstrained in an 8N-dimensional phase space. One additional Lorentz scalar is introduced for an appropriate parametrization of all trajectories, defining also the connection to the proper times of the individual interacting nucleons. The creation of particles, for which Hamiltonian dynamics does not provide a generic mechanism, is incorporated phenomenologically as a ``perturbative'' process. We describe heavy ion collisions as a sequence of two-particle reactions without any additional fit to experimental data. Pion yield and mass spectra of the heavy fragments agree reasonably well with BEVALAC results. We do not compare to experimental high energy data because the employed mechanism of particle production is certainly improper in that regime. However, the numerical results give an impression on how the reaction evolves.