Relative Ion Intensities Research Articles

Improved data-dependent acquisition for untargeted metabolomics using gas-phase fractionation with staggered mass range.

The inherently limited MS2 rate of the mass spectrometer is still restricting the performance of data-dependent acquisition (DDA) for untargeted metabolomics. When dealing with the complex metabolome ocean, top-n-based DDA is just scratching the surface as only a small fraction of the ions could be selected for MS2. Here, we report an improved DDA method for untargeted metabolomics using gas-phase fractionation with staggered mass range (sGPF). Unlike the single m/z segment for conventional GPF, the m/z segments for sGPF were narrower, multiplex, and discrete to allow more homogeneous selection of precursor ions in low, medium, and high m/z ranges. This was achieved indirectly by predefining an inclusion list containing multiple discontinuous m/z ranges. Five fraction levels (2, 4, 6, 8, and 10) and two staggering strategies (staggered wide and narrow subsegments (sGPFa and sGPFb)) were compared for characterizing the human urinary metabolites. For both targeted and untargeted comparison, the highest MS2 coverage was obtained by sGPFb8. Targeted comparison of 60 metabolites indicated sGPFb performed the best for 2, 4, 6, and 8 fractions with an increased MS2 triggering rate of 15.0-36.6% over GPF and 6.6-11.7% over sGPFa. For untargeted screening of phase II metabolites and carboxylates, the best performance achieved by sGPFb8 exhibited a 46.9% increase over GPF8 with the increase evenly distributed in glucuronides (54 vs 38), sulfates (55 vs 41), and carboxylates (31 vs 16). Such superiority of sGPF over GPF is mainly due to the reduced number of concurrent precursor ions and increased relative ion intensity ranks.

Quantification of Sugar Epimers in Polygalactomannans by ESI-MS/MS

Polygalactomannans (PGMs) represent an important family of polysaccharides. They are obtained from the endosperm of leguminous plant seeds and are employed in a growing number of industrial applications as thickening agents and rheology modifiers. Each PGM has a typical mannose/galactose (M/G) ratio (from ~1 to ~5), which determines its solubility, viscosity and other physico-chemical properties. Complex polysaccharides are commonly characterized by proton nuclear magnetic resonance (1H-NMR), whereas mass spectrometry (MS) has not yet seen wide application. In this work, a new quantification method for sugar epimers in PGMs is presented, based on the characteristic fragmentation patterns of mannose and galactose in tandem MS (MS/MS) analyses. Standard galactose and mannose have been analyzed and fragmented in negative-ion mode on a hybrid quadrupole/time-of-flight (qTOF) mass spectrometer equipped with a nano electrospray ionization (ESI) source. The MS/MS spectra indicate accumulation of the same fragments for the two monosaccharides, but significant and reproducible differences in the relative intensities of the product ions. Known mixtures of mannose and galactose have been analyzed by the same procedure to test the applicability of the method for quantification purposes. The resulting peak intensities over the entire MS/MS spectrum can be deconvoluted as a linear combination of the signals from pure mannose and galactose standards, obtaining reliable and reproducible quantification of the epimers. The method has been applied to the characterization of hydrolysis products of PGMs from different species of leguminous plants, such as guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpinia spinosa), in order to assess specific susceptibility to hydrolysis conditions.

Approach to the study of flavone di-C-glycosides by high performance liquid chromatography-tandem ion trap mass spectrometry and its application to characterization of flavonoid composition in Viola yedoensis.

The mass spectrometric (MS) analysis of flavone di-C-glycosides has been a difficult task due to pure standards being unavailable commercially and to that the reported relative intensities of some diagnostic ions varied with MS instruments. In this study, five flavone di-C-glycoside standards from Viola yedoensis have been systematically studied by high performance liquid chromatography-electrospray ionization-tandem ion trap mass spectrometry (HPLC-ESI-IT-MS(n)) in the negative ion mode to analyze their fragmentation patterns. A new MS(2) and MS(3) hierarchical fragmentation for the identification of the sugar nature (hexoses or pentoses) at C-6 and C-8 is presented based on previously established rules of fragmentation. Here, for the first time, we report that the MS(2) and MS(3) structure-diagnostic fragments about the glycosylation types and positions are highly dependent on the configuration of the sugars at C-6 and C-8. The base peak ((0,2) X1 (0,2) X(2)(-) ion) in MS(3) spectra of di-C-glycosides could be used as a diagnostic ion for flavone aglycones. These newly proposed fragmentation behaviors have been successfully applied to the characterization of flavone di-C-glycosides found in V. yedoensis. A total of 35 flavonoid glycosides, including 1 flavone mono-C-hexoside, 2 flavone 6,8-di-C-hexosides, 11 flavone 6,8-di-C-pentosides, 13 flavone 6,8-C-hexosyl-C-pentosides, 5 acetylated flavone C-glycosides and 3 flavonol O-glycosides, were identified or tentatively identified on the base of their UV profiles, MS and MS(n) (n = 5) data, or by comparing with reference substances. Among these, the acetylated flavone C-glycosides were reported from V. yedoensis for the first time.

Serine effects on collision-induced dissociation and photodissociation of peptide cation radicals of the z+●-type

The serine residue displays specific effects on the dissociations of peptide fragment cation-radicals of the z+• type which are produced by electron transfer dissociation. Energy-resolved collision-induced dissociation (ER-CID), time-resolved infrared multiphoton dissociation (TR-IRMPD), and single-photon UV photodissociation at 355 nm revealed several competitive dissociation pathways consisting of loss of OH radical, water, and backbone cleavages occurring at N-terminal and C-terminal positions relative to the serine residue. The activation modes using slow-heating and UV photon absorption resulted in different relative intensities of fragment ions. This indicated that the dissociations proceeded through several channels with different energy-dependent kinetics. The experimental data were interpreted with the help of electron structure calculations that provided fully optimized structures and relative energies for cis and trans amide isomers of the z4+• ions as well as isomerization, dissociation, and transition state energies. UV photon absorption by the z4+• ions was due to Cα-radical amide groups created by ETD that provided a new chromophore absorbing at 355 nm.

Fragmentation Characteristics and Utility of Immonium Ions for Peptide Identification by MALDI-TOF/TOF-Mass Spectrometry

Abstract One of significant characteristics of matrix-assisted laser desorption/ionization tandem time-of-light mass spectrometry (MALDI-TOF/TOF-MS) high-energy collision induced dissociation (CID) is to produce abundant immonium (IM) ions that can offer a wealth of information for peptide composition. However, MALDI-TOF/TOF is generally used for routine protein identification based on database search or de novo sequencing combined with chemical derivation. Consequently, the characteristics of IM ions may not be fully explored and utilized. Here, a total of 239 MS/MS spectra were used to explore the fragmentation features of IM ions with MALDI TOF/TOF spectrometry and their application for peptide identification. IM ion signals for 14 kinds of amino acids including His with a positive rate of more than 50% were observed. The results indicated that the chemical nature of the amino acids and position effect were two main factors that affect the intensity of fragment ions. In addition, false positive IM ions were mainly derived from Arg, Lys, Leu and Ile residues or mixture peptides. Besides the compositional information, partial sequence information was also obtained by comparison of the relative intensity of IM ions. These findings are helpful when performing manual interpretations and can help improving current peptide search algorithms.

Lignification of ray parenchyma cells in the xylem of Pinus densiflora. Part I: Microscopic investigation by POM, UV microscopy, and TOF-SIMS

Abstract The lignification process from sapwood (sW) to heartwood (hW) in ray parenchyma cells (Pray) of Pinus densiflora has been analyzed by means of ultraviolet (UV) microscopy, acetyl bromide (CH3COBr) lignin determination, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The cell wall layers were localized by polarized optical microscopy (POM). POM revealed that Pray have almost no secondary wall in sW and have only the outer layer of secondary wall (S1) in the transition zone (TZ) and hW. UV microscopic observations indicated that the secondary wall of Pray, which is in contact with ray tracheids (Trray), begins to lignify in sW, while the secondary wall of Pray, which is not in contact with Trray, is partially lignified in the TZ. The secondary wall of both types of Pray is completely lignified in hW. The CH3COBr lignin content in sW is slightly lower than that in hW. In the TOF-SIMS measurements, the relative intensities of the secondary ions of guaiacyl-lignin (G-lignin) in the rays in sW are significantly lower than those in hW.

Study of surface multicrystalline substrates silicon saturated aqueous by mass spectroscopy

Hydrogen-saturated surfaces of multicrystalline silicon substrates Baysix with porous silicon, used in the photoelectric converters production were studied using mass-spectrometry methods. Hydrogen saturation was carried out by electrochemical hydrogenation of porous silicon on p-type multicrystalline silicon substrates Baysix with resistivity 1...10 ohm・sm. Comparison of multicrystalline silicon samples surfaces was carried out before and after hydrogenation in the electrolyte, based on hydrofluoric acid, both in the secondary ions spectra and in the image of elements distribution on the surface (mode of ion microprobe and mass-spectral ion microscope). 2D-ion images of the multicrystalline substrate surface were obtained on the mass-spectrometer TOF5 SIMS using the current of hydrogen secondary ions H+ and molecular SiH2+. A number of samples were measured, depending on the electrochemical treatment time in the electrolyte. Analysis of hydrogenation modes, studied by the relative current intensity of the hydrogen secondary ions H+ of the surface of multicrystalline silicon sample Baysix shows that hydrogenation turns into saturation after two hours of treatment. Dynamic etching mode on the mass-spectrometer TOF5 SIMS with determining the hydrogen amount from the surface deep into the studied sample shows that hydrogen concentration, determined by the intensity of secondary ions H+ is higher on the surface and monotonously reduces deep into the sample.

Excitation and ionic fragmentation of the carvone molecule (C10H14O) around the O 1s edge

The electronic excitation and associated ionic dissociation of the carvone molecule have been studied around the oxygen 1s edge, using synchrotron radiation and time-of-flight techniques. Photoabsorption spectrum (total ion yield) and mass spectra have been obtained in the range between 520 and 545eV. For the sake of comparison, carvone mass spectra have also been obtained following valence (21.21eV) and core (carbon 1s) ionization. Fragmentation of the molecule is seen to be greatly enhanced following core excitation. Around the oxygen 1s edge, we observe an extensive fragmentation of the molecular skeleton, as exemplified by the appearance of several previously unreported ions: H+, H2+, CH+, CH2+ and CH3+, which are not formed at low energies. A maximum is observed at 536eV photon energy in the relative intensity of the oxygen-containing ions O+, O2+ and OH+, as an evidence for the existence of site-selective fragmentation of the carvone molecule excited around the O 1s edge. Absolute values for the photoionization and photodissociation cross sections were estimated using the molecular additive rule.

Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-013-0770-2) contains supplementary material, which is available to authorized users.

Amino and Acetamide Functional Group Effects on the Ionization and Fragmentation of Sugar Chains in Positive-Ion Mass Spectrometry

To elucidate the influence of amino (-NH2) and acetamide (-NHCOCH3, -NAc) groups in sugar chains on their ionization and fragmentation, cycloamyloses (cyclodextrins, CyDs) and lacto-oligosaccharide are analyzed by MALDI TOF/TOF and ESI Q-TOF mass spectrometry. CyD derivatives substituted by amino or acetamide groups are ideal analytes to extract the function group effects, which are amino-CyD with one hexosamine (HexNH2) and acetamide-CyD with one N-acetyl hexosamine (HexNAc). Interestingly, the relative ion intensities and isotope-like patterns in their product ion spectra depend on the functional groups and ion forms of sugar chains. Consequently, the results indicate that a proton (H+) localizes on the amino group of the amino sugar, and that the proton (H+) induces their fragmentation. Sodium cation (Na+) attachment is independent from amino group and exerts no influence on their fragmentation patterns in amino group except for mono- and disaccharide fragment ions because there is the possibility of the reducing end effect. In contrast, a sodium cation localizes much more frequently on the acetamide group in acetamide-CyDs because the chemical species with HexNAc are stable. Thus, their ions with HexNAc are abundant. These results are consistent with the fragmentation of lacto-neo-N-tetraose and maltotetraose, suggesting that a sodium cation generally localizes much more frequently on the acetamide group in sugar chains. Graphical abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-013-0739-1) contains supplementary material, which is available to authorized users.

Simultaneous detection of 22 toxic plant alkaloids (aconitum alkaloids, solanaceous tropane alkaloids, sophora alkaloids, strychnos alkaloids and colchicine) in human urine and herbal samples using liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method for simultaneous detection of 22 toxic plant alkaloids, including aconitum alkaloids and their hydrolyzed products (aconitine, hypaconitine, mesaconitine, yunaconitine, crassicauline A, benzoylaconine, benzoylmesaconine, benzoylhypaconine, deacetylyunaconitine, deacetylcrassicauline A), solanaceous tropane alkaloids (atropine, anisodamine, scopolamine, anisodine), sophora alkaloids (matrine, sophoridine, oxymatrine, cytisine, N-methylcytisine), strychnos alkaloids (brucine, strychnine) and colchicine, in herbal and urine samples was developed and validated. Following sample preparation by liquid-liquid extraction, chromatographic separation was achieved on Eclipse XDB C8 column. Identification was based on two multiple reaction monitoring transitions and the relative ion intensity. Method selectivity was demonstrated. The limits of detection were 5ng/mL for all analytes, except 50ng/mL for cytisine. The herbal matrix effects ranged from 89% to 118%, whereas the urine matrix effects were between 91% and 109% for all analytes except cytisine (57%) and N-methylcytisine (67%). The urine extraction recovery ranged from 74% to 110% for all analytes, except cytisine (15%) and oxymatrine (30%). With the good extraction efficiency of the other major sophora alkaloids, the relatively low extraction recovery of the minor sophora alkaloids cytisine and oxymatrine did not affect identification of sophora alkaloids as a group. Carry-over was minimal at less than 0.1%. The method was successfully applied in analysis of 170 cases of suspected herbal poisoning, with aconitum alkaloids, sophora alkaloids, solanaceous tropane alkaloids, and strychnos alkaloids being detected in 53, 42, 18, and 6 cases, respectively.

Determination of pesticide residues and related compounds in water and industrial effluent by solid-phase extraction and gas chromatography coupled to triple quadrupole mass spectrometry

Pollution of drinking water supplies from industrial waste is a result of several industrial processes and disposal practices, and the establishment of analytical methods for monitoring organic compounds related to environmental and health problems is very important. In this work, a method using solid-phase extraction (SPE) and gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS) was developed and validated for the simultaneous determination of pesticide residues and related compounds in drinking and surface water as well as in industrial effluent. Optimization of the method was achieved by using a central composite design approach on parameters such as the sample pH and SPE eluent composition. A single SPE consisting of the loading on a polymeric sorbent of 100 mL of sample adjusted to pH 3 and elution with methanol/methylene chloride (10:90, v/v) permitted the obtaining of acceptable recoveries in most cases. The concentration factor associated with sensitivity of the chromatographic analysis permitted the achievement of the method limit of detection values between 0.01 and 0.25 μg L(-1). Recovery assays presented mean recoveries between 70 and 120% for most of the compounds with very good precision, despite the different chemical nature of the compounds analyzed. The selectivity of the method, evaluated through the relative intensity of quantification and qualification ions obtained by GC-QqQ-MS/MS, was considered adequate. The developed method was finally applied to the determination of target analytes in real samples. River water and treated industrial effluent samples presented residues of some compounds, but no detectable residues were found in the drinking water samples evaluated.

Ionic fragmentation of a CH4molecule induced by 10-keV electrons: Kinetic-energy-release distributions and dissociation mechanisms

The dynamics of ionic fragmentation of CH${}_{4}$ molecules under impact of 10-keV electrons has been studied. The technique of recoil ion momentum spectroscopy is employed to obtain information about the kinetic energy release and the dissociation mechanisms of different pathways arising from the fragmentation of a CH${}_{4}$ dication. The results show that there are altogether eight dissociation pathways that arise from the complete and the incomplete Coulomb explosions of the CH${}_{4}$${}^{2+}$ molecular ions. The kinetic energy release for these pathways is compared with earlier data from the literature for the impact of different charged particles, photons, and their impact energies. The present results indicate that mostly the lower electronic states of CH${}_{4}$${}^{2+}$ are involved for the observed dissociation channels. Also, the dissociation mechanisms associated with these channels are suggested and discussed. Further, we have also estimated the relative ion intensities of different channels of fragmentation of CH${}_{4}$ dication produced under impact of considered energy of electrons.

Development and validation of a QuEChERS based liquid chromatography tandem mass spectrometry method for the determination of multiple mycotoxins in spices

A reliable and rapid method for the determination of multiple mycotoxins was developed using a QuEChERS (quick, easy, cheap, effective, rugged and safe) based extraction procedure in highly pigmented and complex spice matrices, namely red chilli (Capsicum annum ssp.), black and white pepper (Piper nigrum ssp.). High-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) was used for the quantification and confirmation of 17 chemically diversified mycotoxins. Different extraction procedures were studied and optimized in order to obtain better recoveries. Mycotoxins were extracted from the hydrated spices using acidified acetonitrile (1% formic acid), followed by partitioning with NaCl and anhydrous MgSO4; excluding the use of dispersive-solid phase extraction. Significant matrix effect was compensated using the matrix matched calibration curves. Electrospray ionization at positive mode was applied to simultaneously detect all the mycotoxins in a single run time of 20min. Multiple reaction monitoring mode, choosing at least two abundant fragment ions per analyte was applied. Coefficients of determination obtained were in the range of 0.9844–0.9997. Recoveries (ranging from 75% to 117%) were in accordance with the performance criteria required by the European Commission. Intra-day reproducibility ranged from 4% to 22% for most of the mycotoxins. The limit of quantification ranged from 2.3 to 146μgkg−1. The validated method was finally applied to screen mycotoxins in ten of each spice matrix. Aflatoxins, ochratoxin, fumonisins, sterigmatocystin and citrinin were among the detected analytes. Positive findings were further confirmed using relative ion intensities. The potentiality of the method to be used for confirmatory purposes according to Commission Decision 2002/657/EC was assessed.

Correlations of ion structure with multiple fragmentation pathways arising from collision‐induced dissociations of selected α‐hydroxycarboxylic acid anions

Under conditions of collision-induced dissociation (CID), anions of α-hydroxycarboxylic acids usually fragment to yield the distinctive hydroxycarbonyl anion (m/z 45) and/or the complementary product anion formed by neutral loss of formic acid (46 u). Further support for the known two-step mechanism, involving an ion-neutral complex for the formation of the hydroxycarbonyl anion from the carboxyl group, is herein provided by tandem mass spectrometric results and density functional theory computations on the glycolate, lactate and 3-phenyllactate ions. A fourth, structurally related α-hydroxycarboxylate ion, obtained by deprotonation of mandelic acid, showed only loss of carbon dioxide upon CID. Density functional theory computations on the mandelate ion indicated that similar energy inputs were required for a direct, phenyl-assisted decarboxylation and a postulated novel rearrangement to a carbonate ester, which yielded the benzyl oxide ion upon loss of CO2. Rearrangement of the glycolate ion led to expulsion of carbon monoxide, whereas the 3-phenyllactate ion showed the loss of water and formation of the benzyl anion and the benzyl radical as competing processes. The fragmentation pathways proposed for lactate and 3-phenyllactate are supported by isotopic labeling. The relative computed energies of saddle points and product ions for all proposed fragmentation pathways are consistent with the energies supplied during CID experiments and the observed relative intensities of product ions. The diverse reaction pathways characterized for this set of four α-hydroxycarboxylate ions demonstrate that it is crucial to understand the effects of structural variations when attempting to predict the gas-phase reactivity and CID spectra of carboxylate ions.

Analyses of the Iridoid Glucoside Dimers in Paederia scandens Using HPLC–ESI–MS/MS

Many dimers consisting of structurally similar monomers are difficult to be identified even using NMR. Rapid structural identification of iridoid glycoside dimers, especially isomeric dimers in a complicated matrix, remains desirable. To develop a rapid, sensitive analytical method for structural elucidation of trace iridoid glycoside isomers in a complicated extract. Three isomeric iridoid glucoside dimers, paederoscandoside, saprosmoside E and saprosmoside D, were isolated and further analysed by electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) in positive-ion mode. Energy-resolved mass spectrometry (ERMS) was used to provide information on the relative intensity of ions versus collision energy. The crude extract of Paederia scandens was analysed by HPLC-ESI-MS/MS. The relative abundance of product ions in the MS/MS spectra from ammonium adduct ions varied greatly for the three isomers. The energy-resolved experiments enhanced differences among the three isomers. A total of 13 iridoid glucoside dimers (three groups of isomers) in the extract of P. scandens were identified or tentatively characterised by using HPLC-ESI-QTOF based on the tandem mass spectra of references. Linkage sites between different hydroxyl groups on the sugar and carboxyl groups for the three groups of isomers are confirmed. The reason for fragmentation differences might be that cleavage of the glycosidic bond accompanies the active H in vicinal hydroxyl rearrangement. The MS method is a useful tool for the analysis of isomers, especially trace isomers in a complicated extract.

Differentiation of Diastereoisomers of Protected 1,2-Diaminoalkylphosphonic Acids by EI Mass Spectrometry and Density Functional Theory

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-012-0556-y) contains supplementary material, which is available to authorized users.

Radical cascades in electron transfer dissociation (ETD) – implications for characterizing peptide disulfide regio-isomers

Direct characterization of peptides with multiple disulfide bonds by mass spectrometry is highly desirable. In this study, electron transfer dissociation (ETD) of peptide disulfide regio-isomers was studied using model peptides containing two intrachain disulfide bonds. ETD provided rich sequence information (c/z ions) even for the backbone region under the coverage of two disulfide bonds. This behavior presented an analytical advantage over low energy collision-induced dissociation (CID) of protonated intact peptide ions, which produced very limited sequence (b/y) ions. Mechanistic studies suggested that the formation of c/z ions under the two disulfide bond covered region resulted from an initial N-Cα bond cleavage, followed by radical cascades to cleave multiple disulfide bonds. The ETD spectra of the disulfide regio-isomers produced similar product ions due to radical cascades; while the relative intensities of the product ions varied, to a certain degree, which could be helpful in distinguishing isomers with overlapping disulfide bonds.

The pH Dependence of Product Ion Spectra Obtained from Precursor Ions with the Same Charge Number in ESI of Carbonic Anhydrase 2

The effect of solvent conditions, including pH, on product ion spectra obtained from precursor ions produced by electrospray ionization (ESI) was examined. Bovine carbonic anhydrase 2 was used as a model protein and the product ions generated by collision induced dissociation of the whole protein were measured under several different solvent conditions (pH 5.0, 3.7, and 0.1% HCOOH (pH 2.6)/MeCN (1/1)). The product ion spectra from precursor ions with the same charge number, the observed m/z values and the relative intensities of the product ions were similar. It therefore appears that the solvent conditions used have no effect on the product ion that is generated. On the other hand, different profiles of the product ion were obtained from precursor ions having different charge numbers. This indicates that the charge number of the precursor ion appears to be a major determinant of the product ion species and its relative intensity in product ion spectra of proteins.

Dissociation mechanisms of excited CH3X (X = Cl, Br, and I) formed via high-energy electron transfer using alkali metal targets

High-energy electron transfer dissociation (HE-ETD) on collisions with alkali metal targets (Cs, K, and Na) was investigated for CH(3)X(+) (X = Cl, Br, and I) ions by a charge inversion mass spectrometry. Relative peak intensities of the negative ions formed via HE-ETD strongly depend on the precursor ions and the target alkali metals. The dependency is explained by the exothermicities of the respective dissociation processes. Peak shapes of the negative ions, especially of the X(-) ions, which comprise a triangle and a trapezoid, also strongly depend on the precursor ions and the target alkali metals. The trapezoidal part of the I(-) peak observed with the Na target is more dominant and much broader than that with the Cs target. This dependence on the targets shows an inverse relation between the peak width and the available energy, which corresponds to the exothermicity assuming formation of fragment pair in their ground internal states. From a comparison of the kinetic energy release value calculated from the trapezoidal shape of I(-) with the available energy of the near-resonant level on the CH(3)I potential energy curve reported by ab initio calculations, the trapezoidal part is attributed to the dissociation to CH(3) + I((2)P(3/2)) via the repulsive (3)Q(1) state of CH(3)I, which is not dominant in the photo-dissociation of CH(3)I. The observation of trapezoid shape of the CH(2)I(-) peak with the Cs target indicates spontaneous dissociation via repulsive potential from the (3)R(2) Rydberg state, although the correlation between the (3)R(2) Rydberg state and relevant repulsive states has not been reported by any theoretical calculation.