Measuring internal energy deposition in collisional activation using hydrated ion nanocalorimetry to obtain peptide dissociation energies and entropies

The internal energy deposited in both on- and off-resonance collisional activation in Fourier transform ion cyclotron resonance mass spectrometry is measured with ion nanocalorimetry and is used to obtain information about the dissociation energy and entropy of a protonated peptide. Activation of Na⁺(H₂O)₃₀ results in sequential loss of water molecules, and the internal energy of the activated ion can be obtained from the abundances of the product ions. Information about internal energy deposition in on-resonance collisional activation of protonated peptides is inferred from dissociation data obtained under identical conditions for hydrated ions that have similar m/z and degrees-of-freedom. From experimental internal energy deposition curves and Rice-Ramsperger-Kassel-Marcus (RRKM) theory, dissociation data as a function of collision energy for protonated leucine enkephalin, which has a comparable m/z and degrees-of-freedom as Na⁺(H₂O)₃₀, are modeled. The threshold dissociation energies and entropies are correlated for data acquired at a single time point, resulting in a relatively wide range of threshold dissociation energies (1.1 to 1.7 eV) that can fit these data. However, this range of values could be significantly reduced by fitting data acquired at different dissociation times. By measuring the internal energy of an activated ion, the number of fitting parameters necessary to obtain information about the dissociation parameters by modeling these data is reduced and could result in improved accuracy for such methods.     

Boundary effects and collisional activation in a quadrupole ion trap

The effects of storing ions at different values of the stability parameters a_z and q_zwere studied in a quadrupole ion trap, using helium or argon as buffer. A region was localized near the boundaries of the stability diagram in which the ions experience an increase in their kinetic energy. This is reflected in the occurrence both of fragmentation due to collisional activation and of a certain extent of ion loss due to unstable trajectories. The results of this excitation, referred to as ‘boundary effects,’ depend on the specific q_zat which the ion storage is performed and on the buffer gas used, and point to a simpler means of obtaining tandem mass spectra with the ion trap without the need to apply resonant tickle voltages between the end -cap electrodes.     

Characterization of erythromycin analogs by collisional activated dissociation and infrared multiphoton dissociation in a quadrupole ion trap

The effectiveness of two activation techniques, collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), is compared for structural characterization of protonated and lithium-cationized macrolides and a series of synthetic precursors in a quadrupole ion trap (QIT). Generally, cleavage of the glycosidic linkages attaching the sugars to the macrolide ring and water losses constitute the major fragmentation pathways for most of the protonated compounds. In the IRMPD spectra, a diagnostic fragment ion assigned as the desosamine ion is a dominant ion that is not observed in the CAD spectra because of the higher m/z limit of the storage range required during collisional activation. Activation of the lithium-cationized species results in new diagnostic fragmentation pathways that are particularly useful for confirming the identities of the protecting groups in the synthetic precursors. Multi-step IRMPD allows mapping of the fragmentation genealogies in greater detail and supports the proposed structures of the fragment ions.     

Ion internal temperature and ion trap collisional activation: protonated leucine enkephalin

Protonated leucine enkephalin has been used as a prototypical high-mass ion to yield a quantitative estimate of the relationship between the amplitude of the resonance excitation voltage used in an ion trap collisional activation experiment, and the internal temperature to which an ion can be elevated over the bath gas temperature. The approach involves the measurement of the ion dissociation rate as a function of resonance excitation voltage, and the correlation of dissociation rate with ion internal temperature. The relatively high ion trap dissociation rates observed under typical resonance excitation conditions preclude the direct application of the Arrhenius equation to derive internal temperatures. An empirical determination of the relationship between ion internal temperature and dissociation rate over the rate range of interest here was made via the systematic variation of bath gas temperature. The data suggest a very nearly linear relationship between ion internal temperature and resonance excitation voltage, at least under conditions in which ion ejection is minimal. It is shown that protonated leucine enkephalin ions can be elevated by about 357 K over the bath gas temperature using a monopolar resonance excitation voltage of 540 mV p − p(q_z = 0.163) without significant ion ejection. It is also demonstrated that ion internal temperature can be readily increased by increasing the bath gas temperature, by accelerating the ions in the presence of a room temperature bath gas (i.e. conventional ion trap collisional activation), or by a combination of the two approaches.     

Quantitation of isomeric ion mixtures using collisional activation mass spectra

Recently Bass and Bowers have criticized methods for calculation of isomeric composition of gaseous ions based on mass spectra from collisionally activated dissociations (CAD). They fault values from this laboratory on the proportion of benzyl and tropylium ions calculated by two methods from product ion abundances. However, a careful remeasurement of the relevant yields of CAD product ions shows that neither calculation method affected the result outside experimental error, as the ion yields for at least 14 higher energy dissociations are insensitive to both isomeric identity and internal energy. Reducing the [C₇H₇]⁺ internal energy by 1.5 eV does give a 25% reduction in the ion yield for the lowest energy process, supporting the accepted recommendation that these be omitted for quantitative spectral comparisons. A calculation error was made in one of our previous reports on [C₇H₇]⁺, and corrected isomeric composition values are presented. The disparate values from recalculation by Bass and Bowers of other data from this laboratory on [C₂H₄O]^+˙ ions are shown here to be consistent with the large experimental error of those measurements; the medians of their values are actually near the values originally reported. In direct contrast to their assertions, we find no evidence that previous calculation methods have produced misleading conclusions, or that the assumption of linear superposition of CAD spectra in ion mixtures has yielded unreliable results.     

Pulsed helium introduction into a quadrupole ion trap for reduced collisional quenching during infrared multiphoton dissociation of electrosprayed ions

Previous infrared multiphoton dissociation (IRMPD) experiments utilizing a quadrupole ion trap mass spectrometer yielded limited photodissociation efficiencies. Helium buffer gas continuously infused into the analyzer region at pressures of typically 1 x 10(-3) Torr to improve ion trap performance can collisionally quench photoexcited ions during the IRMPD process. Photodissociation experiments have indicated that uncorrected pressures below 2 x 10(-5) Torr are necessary to avoid collisional deactivation of photoexcited ions. This paper describes IRMPD in the quadrupole ion trap at reduced pressures utilizing a dual-pulsed introduction of helium buffer gas incorporated into the ion trap scan function. The pulsed introduction of helium buffer gas before ion injection allows the efficient trapping of ions injected from an electrospray source and the removal of helium before laser irradiation. A second pulse of helium directly before ion detection improves the intensity of the ion signal. The use of this dual-pulsed inlet of helium for improved IRMPD is demonstrated with the carbohydrate antibiotics neomycin and erythromycin. Copyright 2000 John Wiley & Sons, Ltd.     

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.     

Boundary-effect activated dissociation in ion trap tandem mass spectrometry

The potential of boundary-effect activated dissociation (BAD) in ion trap tandem mass spectrometry (MS/MS) is discussed. Several classes of compounds were investigated and the BAD product ion tandem mass spectra were compared with those from collisionally activated dissociation (CAD) where an auxilliary r.f. ‘tickle’ voltage is used. The energy deposition in BAD MS/MS is generally lower than that for optimized CAD, but the experiments are easier to perform. An example of the BAD MS/MS of C₁₀ alkylbenzenes and benzothiophene in diesel fuel using gas chromatographic introduction and predicted r.f. and d.c. voltages is presented.     

Ion trap collisional activation of the deprotonated deoxymononucleoside and deoxydinucleoside monophosphates

Deoxymononucleoside and deoxydinucleoside monophosphate anions formed by electrospray have been subjected to ion trap collisional activation. The threshold for decomposition via loss of base is significantly lower for the deoxymononucleoside 3′-monophosphates than for the corresponding 5′-monophosphates, which indicates that the presence of a charged 3′ phosphate group facilitates base loss. The behavior of the bases among each class of isomers shows slight variation in threshold and tandem mass spectrometry efficiency with tile notable exception of 2′-deoxyguanosine 5′-monophosphate. This ion is exceptionally stable toward decomposition via base loss, which reflects a strong hydrogen bonding interaction between the base and the phosphate group. All dinucleotides fragment via similar mechanisms, but the propensity for neutral base loss relative to loss of a charged base is highly dependent on the identities of both the 5′ and 3′ bases. The behavior of the dinucleotides under collisional activation conditions supports the proposal that base loss proceeds via a proton-bound dimer intermediate in which loss of the charged base directly competes with loss of the neutral base. Application of the kinetic method allows for quantitative predictions of the differences of the gas-phase acidities of the dimer components.     

Orthogonal trap time-of-flight mass spectrometer using a collisional damping chamber

We report a new hybrid mass spectrometer, which is a combination of a quadrupole ion trap and an orthogonal time-of-flight (TOF) mass spectrometer. This new configuration consists of a collisional-damping chamber (CDC) inserted between an MSn-capable ion trap and a high-mass-accuracy orthogonal-TOF mass spectrometer. Because the CDC converted an ion packet into an energy-focused and quasi-continuous beam, a high mass resolution of over 10,000 and a high mass accuracy of better than 3 ppm were achieved. Moreover, the ratio of the maximum detectable m/z to the minimum detectable m/z, which is referred to here as the mass window, was improved to more than 10.     

Photodissociation and collisional cooling of rhodamine 575 cations in a quadrupole ion trap

The photodissociation of rhodamine 575 cations held in a quadrupole ion trap is studied using 514 nm light as a function of buffer gas pressure, irradiation time, and laser fluence. The laser-induced photodissociation decays of rhodamine ions have lifetimes on the order of seconds for the range of pressures and powers investigated and exhibit strong nonlinear pressure dependence. Dissociation mechanisms are considered that involve the sequential absorption of multiple photons and several collisional deactivation steps.     

Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.     

Relative binding energies of gas-phase pyridyl ligand/metal complexes by energy-variable collisionally activated dissociation in a quadrupole ion trap

The relative binding energies of a series of pyridyl ligand/metal complexes of the type [M(I)L(2)](+) and [M(II)L(3)](2+) are investigated by using energy-variable collisionally activated dissociation in a quadrupole ion trap mass spectrometer. The pyridyl ligands include 1,10-phenanthroline and various alkylated analogues, 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine, and 2,2':6',2' '-terpyridine, and the metal ions include cobalt, nickel, copper, zinc, cadmium, calcium, magnesium, lithium, sodium, potassium, rubidium, and cesium. The effect of the ionic size and electronic nature of the metal ion and the polarizability and degree of preorganization of the pyridyl ligands on the threshold activation voltages, and thus the relative binding energies of the complexes, are evaluated. Correlations are found between the binding constants of [M(II)L(3)](2+) complexes in aqueous solution and the threshold activation voltages of the analogous gas-phase complexes determined by collisionally activated dissociation.     

Energy-resolved collisional activation of dimethyl phosphonate and dimethyl phosphite ions in a quadrupole ion trap and a triple quadrupole mass spectrometer

Collision-activated dissociation spectra of dimethyl phosphonate and dimethyl phosphite ions were measured as a function of the amplitude of a supplementary AC voltage applied across the end-caps of an ion-trap mass spectrometer. These spectra yield breakdown graphs which bear a close resemblance to those obtained by varying collision energy in a triple-quadrupole mass spectrometer operating under multiple-collision conditions. Variation in the time of excitation at the resonance frequency provides an alternative route to breakdown graphs. The results demonstrate that energy deposition occurs via multiple activating collisions in the ion trap. Maximum energy deposition observed is somewhat smaller under normal operating conditions in the ion trap than in the triple-quadrupole mass spectrometer.     

Gas-phase stability of double-stranded oligodeoxynucleotides and their noncovalent complexes with DNA-binding drugs as revealed by collisional activation in an ion trap

The intrinsic (gas-phase) stabilities of duplex, self-complementary oligonucleotides were measured in a relative way by subjecting the duplex precursor ions to increasing amounts of collision energy during the collisional-activated decomposition (CAD) events in an ion-trap mass spectrometer. The results are displayed as a dissociation profile, an s-shaped curve that shows the dependence of the relative abundance of the duplex on the applied collision energy. The total number of charges, the total number of base pairs, and the location of the high proton-affinity bases (i.e., G and C) are the main factors that affect the intrinsic stability of the duplex oligonucleotides. If the charge state is the same, the stability, as measured as a half-wave collision energy, E1/2, correlates well with the total number of H bonds for the duplex. The intrinsic stabilities of noncovalent complexes between duplex oligonucleotide and some DNA-binding drugs were also measured by using the newly developed method. Although duplexes are stabilized in the gas phase when they bind to drug molecules, correlations between gas-phase stabilities and the solution-binding affinities have not yet been obtained. Complexes in which the drug is bound in the minor groove must be joined tightly because they tend to dissociate in the gas phase by breaking covalent bonds of the oligonucleotide to give base loss and small sequence-ion formation. Complexes in which the drug is known to favor intercalation dissociate by breaking weak, noncovalent bonds to form single-stranded oligonucleotides although cleavage of covalent bonds of the oligonucleotide also occurs.     

Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74–95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA^phe; rather, the mass was consistent with tRNA^Phe bearing an incomplete 3′-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5′- and 3′-termini of the molecule could be used to identify the major component as tRNA^phe-3′adenosine (without 3′-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA^Phe variant and the intact tRNA^Phe was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA^Phe-3′adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.     

Mass spectral fragmentation pathways in RDX and HMX. A mass analyzed ion kinetic energy spectrometric/collisional induced dissociation study

A collisional induced dissociation study of 1,3,5-trinitro-1,3,5 triazacyclohexane (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX) was carried out using mass analyzed kinetic energy spectrometry. High resolution mass spectra and mass analyzed ion kinetic energy/collisional induced dissociation spectra of RDX and HMX were recorded in the electron impact, chemical ionization and negative ion chemical ionization modes. Fragmentation pathways of the compounds investigated were determined in all three modes of ionization. It was found that a major part of the fragment ions in RDX and HMX originate from formation of the aduct ions [M+NO]⁺ and [M+NO₂]⁺ in electron impact and chemical ionization, and from [M+NO]^? and [M+NO₂]^? in negative chemical ionization, followed by dissociation.     

Delineating mechanisms of dissociation for isomeric heparin disaccharides using isotope labeling and ion trap tandem mass spectrometry

Heparin and heparan sulfate (HS) glycosaminoglycans have been identified as important players in many physiological as well as pathophysiological settings. A better understanding of the biosynthesis and structure of these molecules is critical for further elucidation of their biological function. We have demonstrated the successful use of negative electrospray ionization tandem mass spectrometry in the differentiation of all twelve standard heparin-building blocks, including the potentially important N-unsubstituted disaccharides. Collision induced dissociation of each of the isomeric disaccharides provided unique product ion spectra, useful for identification and quantification of the relative amounts of each isomer present. In the research presented herein, isotopic labeling studies using (18)O and (2)H were used to determine the origins of each of the neutral losses observed in the product ion spectra, and mechanisms of dissociation consistent with the observed data were postulated. The general mechanisms postulated were for the generation of B, Y, and Z ions formed from glycosidic cleavages, as well as A and X ions formed from cross-ring cleavages. The eight isomeric heparin disaccharides all underwent cross-ring cleavage to form (0,2)X(1) and (0,2)A(2) ions, and further experiments suggest that the mechanisms of formation of these ions are through a charge-remote process. The tandem mass spectrometry data presented herein also provide a foundation for further developments towards a practical analysis tool for the structural elucidation of larger, biologically important heparin/HS oligosaccharides by using mass spectrometry.