High-energy collisional activation studied via angle-resolved translational energy spectra of survivor ions.

Angle-resolved translational energy spectroscopy has been applied to Cs4I + (3) ions that survived 8 keV collisions with a range of collision gas targets, including inert gases and deuterium. The experimental data comprise values of the translational energy loss ΔTR as a function of the (laboratory-frame) scattering angle θ R for each collision gas under conditions such that single-collision events dominated the scattering. The values of ΔTR increase with θ R, in accordance with very general expectations. However for any value of θ R, the values of ΔTR for helium and deuterium as targets were almost indistinguishable from one another but were at least five to six times larger than those for neon and all other collision gases. These data have been shown to be consistent with theoretical considerations based upon conservation of energy and linear momentum. Theoretical approaches include the simple "elasticlimit" model, which makes no mechanistic assumptions, and a particular "binary-model" theory, which excludes electronic excitation as a possibility. Both theories are consistent with the experimental data and interpret the surprisingly large values of ΔTR for low-mass targets in terms of large recoil energies of the target required to ensure conservation of momentum. The most likely alternative candidate as sink for ΔTR is internal excitation of the target, but this possibility was excluded in the present work by choosing ΔTR values less than the lowest excitation energies of the inert gas targets. Moreover, such an interpretation cannot explain the similar results obtained using helium and deuterium, which were markedly different from those obtained for all other collision gases.

Characterization of a high-pressure quadrupole collision cell for low-energy collision-indneed dissociation.

A RF-only quadrupole collision cell of new design has been evaluated for use in tandem mass spectrometry experiments as a component of a triple quadrupole mass spectrometer. The new design permits operation at values of collision gas thickness higher by 1 order of magnitude than those used in most cells of this type. When operated at sufficiently high collision gas pressures, the transmission efficiency for precursor ions increases with increasing pressure, often to values greater than those observed in the absence of collision gas. Simultaneously, the attainable resolving power for fragment ions across the entire mass-tocharge ratio range, even for multiply charged precursors, also increases to the point where isomers of a quadruply charged fragment are resolved. The performance of the cell, judged in terms of yields and resolution of fragment ions, has been investigated as a function of the nature and pressure of collision gas, the kinetic energy of the precursor ions that enter the cell, and of the size and charge state of the precursors. The enhanced performance is explicable in terms of a marked deceleration of all ions that emerge from the cell to very low energies, probably a few tens of millielectronvolts, so that the cell effectively acts as an ion source for the second mass filter (fragment ion analyzer) to provide a spectrum of ions of fixed axial energy. The high transmission efficiency appears to arise from a collisional focusing effect analogous to that exploited in three-dimensional RF ion traps. The low axial energies imply that ion transit times through the cell are sufficiently long (several milliseconds) that, in precursor ion experiments where the first mass filter is scanned, a hysteresis effect is observed. This implies that in this operating mode compromises must be sought between scan speed and quality of peak shape. Examples are given of spectra obtained under realistic operating conditions that employ flow injection of samples.

Delayed dissociation spectra of survivor ions from high-energy collisional activation.

Collisional activation (CA) of large ions at kiloelectronvolt energies is accompanied by unexpectedly large losses of translational energy, which vary with the nature of the collision gas. Previous investigations have concentrated upon subsequent fragmentations occurring within a time window covering a few fis immediately following collision, using massanalyzed ion kinetic energy spectrometry. In the present work, survivor ions were selected for specified values of translational energy loss, and their internal energy contents assessed via their subsequent unimolecular fragmentation reactions within a later time window. Beam collimation was also applied when circumstances permitted to impose angular selection, thus minimizing cross talk between effects of collisional scattering and energy dispersion. It was shown that internal excitation of the reactant ion can account for only a small fraction of the observed loss of translational energy. The recoil energy of the target is thus the principal sink for the translational energy loss, since the latter was always chosen to be less than the lowest excitation energy of the target. This conclusion is shown to be consistent with theoretical models of the CA process. The practical implications of these conclusions for CA of large ions at kiloelectronvolt energies are discussed.

Effects of ionization mode on charge-site-remote and related fragmentation reactions of long-chain quaternary ammonium ions.

Comparison of collisionally activated fragment spectra of long-chain quaternary ammonium ions, formed by liquid-assisted secondary ion mass spectrometry (LSIMS) and electrospray ionization (ESI), shows the latter are dominated by radical cations while the former yield mainly even-electron charge-site-remote (CSR) fragments, similar to the report for different precursors by Cheng et al., J. Am. Soc. Mass Spectrom. 1998, 9, 840. Here, mixed-site fragmentation products (formal loss of a radical directly bonded to the nitrogen plus a radical derived from the long chain) are of comparable importance for both ionization techniques. These observations are difficult to understand if the CSR ions are formed by a concerted rearrangement-elimination reaction, since precollision internal energies of the ESI ions are much lower than those of the ions from LSIMS. Alternatively, if one discards the concerted mechanism for high-energy CA, and assumes that the even-electron fragments are predominantly formed via homolytic bond cleavage, the colder radical cations from ESI survive to the detector while the more energized counterparts from LSIMS preferentially lose a hydrogen atom to yield the CSR ions, as proposed by Wysocki and Ross (Int. J. Mass Spectrom. Ion Processes 1991, 104, 179). The present work also attempts to reconcile discrepancies involving critical energies and known structures for neutral fragments.

Rearrangements of doubly charged acylium ions from lysyl and ornithyl peptides.

The study of large-scale rearrangements of [b']2+ ions produced by electrospray ionization of Substance P (Tang et al., Anal. Chem. Vol. 65, p. 2824 (1993)) has been extended to 18 other peptides containing either a lysine or ornithine residue remote from the C-terminus. Evidence for wholesale transfer of one or more residues, from the C-terminus of the [b']2+ precursor to the omega-amino group of the Lys (or Orn) residue, was observed for 12 of the 18 peptides studied. Unfortunately, no rigorous predictive rules, relating features of the peptide sequence to the propensity to undergo such rearrangements, could be discerned although a significant correlation with presence of a proline residue close to the lysine or ornithine on the C-terminal side was apparent. The resulting mass-shifts can complicate derivation of peptide sequences from fragment-ion spectra of [M + 2H]2+ peptide ions, for example, since the cyclized [b']2+ ions responsible for the rearrangements are readily formed as intermediate species in the fragmentation mechanisms.

An investigation of fragmentation mechanisms of doubly protonated tryptic peptides.

Peptides formed as reaction products, of specific hydrolysis of proteins by trypsin, are characterized by a basic residue (Arg or Lys) at the C-terminus, which facilitates formation of abundant [M + 2H]2+ ions under electrospray or ionspray conditions. These doubly charged ions readily dissociate upon collisional activation to y" and b fragment ions which are mass complements of one another. The suggestion that these fragments are formed by direct charge-separation dissociations must contend with the observation that the y" intensities are generally appreciably larger than those of their b counterparts. However, it is shown that this can be accounted for by a greater susceptibility of the b ions to undergo further dissociation to smaller fragments such as immonium ions. In addition no evidence could be found to support alternative mechanisms, including dissociative electron capture, for which equal intensities of the two fragment ion series are not obligatory. Initial protonation at the N-terminus was shown to be required for formation of these [M + 2H]2+ ions via its suppression by mono-acetylation at the N-terminus. These findings, and others concerning formation of [y"']2+ fragments, are consistent with extensions of published mechanisms for formation of b and of y" fragments from singly protonated peptides, via charge-site-induced cleavages and intramolecular proton transfers between nitrogen atoms, respectively.

Ion dissociation reactions induced in a high-pressure quadrupole collision cell.

This work concerns a new high-pressure quadrupole collision cell, designed for triple-quadrupole mass spectrometers. This new collision cell operates at pressures up to 10 mTorr, an order of magnitude higher than conventional cells of this type. Previous investigations have concentrated upon the significant increases in transmission efficiency and in resolving power for fragment ions which result from the use of this new design. The present work reports an investigation into the nature of the dissociation reactions which can be induced by collisions in this high-pressure cell. Charge-site-remote fragmentations of a simple precursor ion were chosen as a test case, and were found to be observable at laboratory collision energies lower by a factor of 4-5 than those found previously to be necessary when using conventional low-pressure quadrupole collision cells. It was also shown that the charge-site-remote reactions were accompanied by the mixed-site-fragmentation reactions described by Tuinman and Cook (J. Am. Soc. Mass Spectrom. Vol. 1, p. 85 (1989)). Ionization of collision gas was observed in the case of xenon. Efforts to observe charge-site-remote fragmentations of peptide ions were marginally successful. Highly basic peptides, which have been problematic for sequencing by low-energy tandem mass spectrometry, did not yield useful fragment-ion spectra in the new cell. The fragmentation behaviour of protonated Leu-enkephalin, for which fragmentation pathways have been thoroughly studied previously, suggested that the observed spectra reflected integration of the fragmentation kinetics over a considerably longer time, thus involving many more reaction steps. These combined observations are considered in terms of a qualitative model based on a rapid decrease of ion kinetic energy during passage through the cell, with much longer residence times than for conventional quadrupole cells.

Fragmentation pathways in electron impact mass spectra of methoxyhalobiphenyls.

The electron impact mass spectra of various methoxyhalobiphenyls have been reinvestigated. Previous findings, concerning the effect of the methoxy group upon the main fragmentation routes, have been confirmed and extended. In addition, studies of unimolecular and collision induced fragmentation of ions, using linked scan techniques, have been made. Such an approach permits the identification of substances with a minimal chromatographic separation prior to mass spectrometry. It has also led to some clarification of the fragmentation pathways leading to the observed cracking patterns.

Fragmentation reactions of multiply-protonated peptides and implications for sequencing by tandem mass spectrometry with low-energy collision-induced dissociation.

The low-energy collision-induced dissociation reactions of a series of multiply-protonated peptides have been investigated by tandem mass spectrometry. It is known that doubly-protonated tryptic peptides undergo facile fragmentation yielding redundant sequence information. The present work has shown that this fortunate circumstance seems likely to be the exception rather than the rule. The presence of additional basic residues, at positions other than the C-terminus, complicates the spectra. The most important such complication discovered in the present work involves wholesale transfer of one or two residues from the C-terminal end of a doubly-charged b fragment to the side chain of a lysine residue located near the N-terminus, resulting in mass shifts of the products of subsequent second-stage fragmentations. Other examples of the participation of the flexible lysine side chain are suggested but could not be confirmed to the same extent. The role of Coulombic repulsion in facilitating fragmentation has been explored via investigations of triply- and quadruply-protonated basic peptides bearing one charge for every three or four amino acid residues. Such species yielded almost no sequence information under low-energy collision conditions, due to the localization of the ionizing protons on highly basic sites rather than on the peptide backbone. It is proposed that collisionally activated mobilization of protons from the basic sites, where they are originally located upon formation, to the backbone is a necessary condition for structurally useful fragmentation to occur. It was not possible, on the basis of the present work, to deduce mechanistic generalizations and predictive schemes which would permit structural interpretations of such fragment spectra for unknown peptides.

Isolation, mass spectrometric characterization, and protein phosphatase inhibition properties of cyclic peptide analogues of gramicidin-S from Bacillus brevis (Nagano strain).

Six substitutional variants of gramicidin-S, present in a commercial extract of Bacillus brevis, have been characterized using a combination of liquid chromatographic/mass spectrometric analysis of the crude extract, plus accurate mass measurements, tandem mass spectrometric investigations, and amino acid analyses, of isolated high-performance liquid chromatographic fractions. Two of these variants were shown previously (Nozaki and Muramatsu, J. Antibiotics 37, 689 (1984)) to involve replacement of one or both Val residues in gramicidin-S by aminobutyric acid, and these findings are confirmed here. One of the four unknown variants corresponds to substitution of one Val residue by Leu, while the other three all involve substitution of one of the Orn residues in gramicidin-S by Lys, or by citrulline (gamma-carbamoyl ornithine), or by gamma-formyl ornithine. The biological activities of the purified fractions were assessed with respect to their in vitro inhibition of protein phosphatases PP1 and PP2A. For both protein phosphatases inhibition levels fell in the micromolar range, about four orders of magnitude higher than IC50 values for other small cyclic peptides such as microcystins and nodularins.

Gas chromatographic/mass spectrometric analysis of specific isomers of polychlorodibenzofurans.

The gas chromatographic/mass spectrometric characteristics of 26 congeners of polychlorinated dibenzofurans, previously characterized by specific synthetic routes and by standard spectroscopic techniques, have been evaluated. The electron impact mass spectra are not particularly isomer-specific, though 2,3,7,8-tetrachlorodibenzofuran is distinguishable on this basis from the three other tetrachloro isomers investigated in this work. Positive ion methane chemical ionization mass spectra do show a greater degree of isomer distinction, and are reasonably reproducible. Electron attachment negative ion spectral characteristics are also presented. Preliminary results on negative ion chemical ionization mass spectra, obtained using methane plus small amounts of oxygen as reagent gas, are reported.

Fast atom bombardment/tandem mass spectrometry of physalaemin-like peptides.

Fragment ion spectra obtained from collision-induced decomposition of protonated molecular ions have been used to determine amino acid sequences of several physalaemin-like peptides which were recently purified from rabbit stomach. This technique was chosen because the peptides were available in microgram quantities and were anticipated to contain pyroglutamate as the blocked N-terminal residue. Such spectra of several synthetic analogs of the naturally occurring peptides were obtained and analyzed to confirm the veracity of this peptide sequencing strategy. In addition, methyl ester derivatives of these synthetic peptides provided a crucial test for the spectral interpretation via the mass shifts thus induced.

Glycerol 1,2-cyclic phosphate in centric diatoms. Observation by 31P NMR in vivo, isolation, and structural determination.

We have isolated and identified glycerol 1,2-cyclic phosphate as the compound responsible for a unique prominent resonance at 19.1 ppm in the 31P NMR spectrum in vivo of four species of centric diatoms, where it is responsible for 15 to 30% of the total signals from organophosphates. This appears to be the first observation of this compound as a major metabolite. It was not detectable in nine other species of algae, including four species of pennate diatoms.

Determination of erythromycin A in salmon tissue by liquid chromatography with ion-spray mass spectrometry.

A reverse-phase liquid chromatography/mass spectrometry (LC/MS) method, incorporating gradient elution, is described for the characterization of residual erythromycin A and its metabolites in salmon tissue. The method uses ion-spray, a mild atmospheric pressure ionization technique which provides an abundant protonated molecule well suited for selected ion monitoring experiments. Tandem mass spectrometry (MS/MS) using collision-induced dissociation was used to provide structural information. The LC/MS method was tested for the analysis of salmon tissue spiked with erythromycin A at levels between 0.01 and 1 p.p.m. A simple extraction and clean-up procedure, slightly modified from that described by Takatsuki et al. (J. Assoc. Off. Anal. Chem. 70, 708 (1987)), was used in this work. Using selected ion and selected reaction monitoring techniques, the LC/MS and LC/MS/MS methods provided detection limits of < 10 and 50 ng g-1, respectively. Confirmatory full-scan LC/MS and LC/MS/MS spectra were obtained at the 0.5 and 1 microgram g-1 levels, respectively. Using a combination of these techniques, the presence of residual erythromycin A was confirmed in the tissue of fish administered medicated feed containing the antibiotic. In addition, several metabolites and degradation products of erythromycin A, including anhydro-erythromycin and N-demethyl-erythromycin, were detected and where possible confirmed by comparison with authentic compounds. Although this analytical method has been shown to afford the necessary sensitivity and precision, application of these techniques to high-throughput quantitative analyses will require development of an improved clean-up procedure and preferably also of a suitable surrogate internal standard.

C60 and C70 fullerene isomers generated in flames. Detection and verification by liquid chromatography/mass spectrometry analyses.

Fullerenes C60 and C70, generated by combustion, have been shown previously to be produced in controlled laminar flames accompanied by other compounds having fullerene-like characteristics. Analysis of these additional compounds by high-performance liquid chromatography, coupled on-line with mass spectrometry has identified them as isomers of the C60 and C70 fullerenes. The newly observed isomers have characteristic UV spectra and are thermally unstable, undergoing conversion to the more stable fullerenes with a half-life of about 1 h in boiling toluene (111 degrees C). Isomers of C60 and C70 fullerenes previously have been studied theoretically, but not observed experimentally. The flame-generated material also contains C60O and C70O compounds, as well as C76 and higher carbon clusters.

Zinc from oyster tissue as causative factor in mouse deaths in official bioassay for paralytic shellfish poison.

Toxicity (extreme weakness, body temperature drop, cyanosis, some slow deaths) in test mice, upon intraperitoneal injection of standard-method paralytic shellfish poison (PSP) extracts of some PSP-free oysters, is consistent with the relatively high levels of zinc in these extracts. As a rough guideline, the threshold for a toxic response corresponds to a drained tissue zinc level of over 900 micrograms/g. The identification of zinc as the substance responsible has been supported by inducing toxicity in control extracts by spiking with nontoxic levels of zinc, and by eliminating toxicity from toxic extracts by chemical removal (precipitation, ion exchange) of metals.