Resonance excitation and dynamic collision-induced dissociation in quadrupole ion traps using higher-order excitation frequencies

Fragmentation of the pentapeptide leucine enkephalin (YGGFL) is accomplished via higher-order resonances combined with simultaneous analysis of low-mass product ions. Two methods of achieving excitation are explored: (1) 0.5 ms resonant excitation at the omega and at Omega-omega secular frequencies of ion motion (where Omega is the radio-frequency (rf) drive frequency) in a manner similar to both pulsed q collision-induced dissociation (PQD) and high amplitude short time excitation (HASTE), and (2) 0.5 ms pulse of the omega or at Omega-omega excitation frequencies when the secular frequency of the ions is quickly swept across resonance conditions (pulsed q dynamic CID, PqDCID). In both methods of excitation, the rf amplitude on the ring electrode is rapidly decreased after excitation, therefore enabling analysis of low-mass product ions. Maximum fragmentation efficiencies of approximately 20% can be obtained with pulsed CID with both regular and high-order frequency excitation, while pulsed DCID offers maximum efficiencies of approximately 12%. All the excitation methods studied offer increased internal energy depositions when compared to conventional CID, as measured by the a4/b4 product ion ratios of leucine enkephalin. These ratios were as high as 13:1 for pulsed CID and 8:1 for PqDCID. Successful mass analysis of the low-mass ions is observed with both pulsed CID and PqDCID. The combined benefit of high internal energy deposition and wider dynamic mass range offers the possibility of increased sequence coverage and the identification of unique internal fragments or high-energy product ions which may provide complementary information to biological applications of conventional CID. This is the first report on deliberate fragmentation of precursor ions at a higher-order component of the ion secular frequency combined with a successful mass analysis of the low-mass ions through pulsed CID and PqDCID.     

High amplitude short time excitation: A method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer

Collision induced dissociation (CID) in a quadrupole ion trap mass spectrometer using the conventional 30 ms activation time is compared with high amplitude short time excitation (HASTE) CID using 2 ms and 1 ms activation times. As a result of the shorter activation times, dissociation of the parent ions using the HASTE CID technique requires resonance excitation voltages greater than conventional CID. After activation, the rf trapping voltage is lowered to allow product ions below the low mass cut-off to be trapped. The HASTE CID spectra are notably different from those obtained using conventional CID and can include product ions below the low mass cut-off for the parent ions of interest. The MS/MS efficiencies of HASTE CID are not significantly different when compared with the conventional 30 ms CID. Similar results were obtained with a two-dimensional (linear) ion trap and a three-dimensional ion trap.     

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.     

Implementation of dipolar direct current (DDC) collision-induced dissociation in storage and transmission modes on a quadrupole/time-of-flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).     

Dynamic collision-induced dissociation (DCID) in a quadrupole ion trap using a two-frequency excitation waveform: I. Effects of excitation frequency and phase angle

This study describes the application of a two-frequency excitation waveform to the end-cap electrodes of a quadrupole ion trap (QIT) during the mass acquisition period to deliberately fragment selected precursor ions. This approach obviates the need for a discrete excitation period and guarantees on-resonant excitation conditions without any requirement for resonant tuning; it is therefore faster than the conventional approach to collision-induced dissociation (CID) in QITs. The molecular ion of n-butylbenzene is used as thermometer molecule to determine the energetics of the new excitation procedure. The excitation waveform, consisting of two closely spaced sinusoidal frequencies, has an interference pattern that displays nodes and crests in the time domain. The energetics (determined by the product ion ratios of 91/92 Th) and CID efficiencies are highly dependent on the excitation amplitude, the relative position of the excitation frequencies in the Mathieu stability diagram, and whether the ions come into resonance during a node or crest of the excitation waveform. Under highly energetic conditions, ratios of 91/92 as large as 15 can be obtained at concomitant CID efficiencies of 10%, indicating internal energies in excess of 10 eV at the time of fragmentation. These extremely high internal energies far exceed the energetics achievable using conventional on-resonance excitation in QITs, indicating that the collisional heating rate is very fast in the new approach. Under less energetic conditions CID efficiencies as high as 70% are possible, which compares favorably with results obtained by conventional on-resonance excitation. Correlation analyses are used to determine the conditions that simultaneously optimize energetic and efficient fragmentation conditions.     

Energetics and efficiencies of collision‐induced dissociation achieved during the mass acquisition scan in a quadrupole ion trap

Using n‐butylbenzene as a test molecule, evidence is provided that fast, efficient or highly energetic collision‐induced dissociation (CID) can be achieved during the mass acquisition ramp of a commercially available quadrupole ion trap (QIT) mass spectrometer. The method of excitation is very similar to axial modulation for mass range extension except that lower amplitude waveforms are used to excite the precursor ions within the trap instead of ejecting them from the trap. ITSIM simulations verify that fast kinetic excitation followed by kinetic‐to‐internal energy transfer occurs on the rapid time‐scale required for the recapture and mass analysis of product ions during the mass acquisition ramp. CID efficiencies larger than 50% can be obtained using this new approach and ratios of Th 91/92 of n‐butylbenzene fragment ions as large as 9 are possible, albeit at significantly reduced efficiencies. These very large ratios indicate an internal energy above 7 eV for the precursor ions indicating that fragmentation of larger ions could also be possible using this new approach. The main benefits of the new method are that no extra time is required for fragmentation or cooling and that on‐resonance conditions are guaranteed because the ions' secular frequencies are swept through the fixed frequency of excitation. Also presented are the effects of experimental variables such as excitation frequency, excitation amplitude and scan rate on the CID efficiencies and energetics. Copyright © 2005 John Wiley & Sons, Ltd.     

Comparison of the effects of ionization mechanism, analyte concentration, and ion “cool-times” on the internal energies of peptide ions produced by electrospray and atmospheric pressure matrix-assisted laser desorption ionization

The propensities of a series of peptide ions produced by both electrospray and atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) to fragment in an ion trap mass spectrometer under various conditions were studied in detail by measuring the extent of fragmentation of precursor ions by collision induced dissociation (CID) as a function of applied resonance excitation RF voltage. For the most basic peptides, the energy required to fragment MH+ ions generated by electrospray exceeded that required to fragment equivalent AP-MALDI ions under identical instrumental conditions; the reverse was observed for a peptide incorporating no basic residues, while peptides of intermediate basicity showed little difference between the ionization methods. This correlation between peptide basicity and the difference in the energy required to induce fragmentation of MH+ ions generated by AP-MALDI and electrospray is attributed primarily to a trend in the internal energies of the ions generated by AP-MALDI (the greater the difference in gas-phase basicities between the matrix and the analyte the greater the internal energy of the analyte ions produced). Furthermore the internal energies of ions produced by AP-MALDI, but not the equivalent ions formed by electrospray, were observed to decrease with decreasing analyte concentration. We attribute this finding to the cooling effect of endothermic dissociation of analyte ion/matrix molecule clusters following the matrix assisted laser desorption step. Time-resolved analyses (measurement of extent of fragmentation of precursor ions by CID as a function of pre-CID "cool times") revealed that cooling periods in excess of 250 ms were required to achieve internal energy equilibrium through cooling collisions with the helium buffer gas. Furthermore, these analyses demonstrated that, even after these extended cooling times, equivalent ions formed by the two ionization techniques showed different propensities to fragment. We conclude that the two different ionization techniques produce ion populations that may differ in their three-dimensional structure.     

Dipolar DC Collisional Activation in a “Stretched” 3-D Ion Trap: The Effect of Higher Order Fields on rf-Heating

Applying dipolar DC (DDC) to the end-cap electrodes of a 3-D ion trap operated with a bath gas at roughly 1 mTorr gives rise to ‘rf-heating’ and can result in collision-induced dissociation (CID). This approach to ion trap CID differs from the conventional single-frequency resonance excitation approach in that it does not rely on tuning a supplementary frequency to coincide with the fundamental secular frequeny of the precursor ion of interest. Simulations using the program ITSIM 5.0 indicate that application of DDC physically displaces ions solely in the axial (inter end-cap) dimension whereupon ion acceleration occurs via power absorption from the drive rf. Experimental data shows that the degree of rf-heating in a stretched 3-D ion trap is not dependent solely on the ratio of the dipolar DC voltage/radio frequency (rf) amplitude, as a model based on a pure quadrupole field suggests. Rather, ion temperatures are shown to increase as the absolute values of the dipolar DC and rf amplitude both decrease. Simulations indicate that the presence of higher order multi-pole fields underlies this unexpected behavior. These findings have important implications for the use of DDC as a broad-band activation approach in multi-pole traps.     

Comparison of collision-induced dissociation and electron-induced dissociation of <Emphasis Type="Italic">singly protonated</Emphasis> aromatic amino acids,cystine and related simple peptides using a hybrid linear ion trap–FT-ICR mass spectrometer

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.     

Establishing low-energy sequential decomposition pathways of leucine enkephalin and its N- and C-terminus fragments using multiple-resonance CID in quadrupolar ion guide

The simultaneous resonant low-energy excitation of leucine enkephalin and its fragment ions was demonstrated in a collision cell of the multipole-quadrupole time-of-flight instrument. Using low-amplitude multiple-resonance excitation CID, we were able to show the exclusive sequential fragmentation of N- and C-terminus fragments all the way to the final fragments--immonium ions of phenylalanine or tyrosine. In this CID mode the single-channel dissociation of each new generation of fragments followed the lowest energy decomposition pathways observable on the time scale of our experiment. Up to six generations of sequential dissociation were carried out in multiple-resonance CID experiments. The direct qualitative comparison of fragmentation of axial-acceleration versus resonant (radial) CID was performed in the same instrument. In both activation methods, fragmentation patterns suggested complex decomposition mechanisms attributable to dynamic competition between sequential and parallel dissociation channels.     

Fragmentation of protonated dipeptides containing arginine. Effect of activation method

The fragmentation reactions of the protonated dipeptides Gly-Arg and Arg-Gly have been studied using collision-induced dissociation (CID) in a quadrupole ion trap, by in-source CID in a single-quadrupole mass spectrometer and by CID in the quadrupole cell of a QqTOF mass spectrometer. In agreement with earlier quadrupole ion trap studies (Farrugia, J. M.; O'Hair, R. A. J., Int. J. Mass Spectrom., 2003, 222, 229), the CID mass spectra obtained with the ion trap for the MH(+) ions and major fragment ions are very similar for the two isomers indicating rearrangement to a common structure before fragmentation. In contrast, in-source CID of the MH(+) ions and QqTOF CID of the MH(+), [MH - NH(3)](+) and [MH <23 HN = C(NH(2))(2)](+) ions provide distinctly different spectra for the isomeric dipeptides, indicating that rearrangement to a common structure has not occurred to a significant extent under these conditions even near the threshold for fragmentation in the QqTOF instrument. Clearly, under normal operating conditions significantly different fragmentation behavior is observed in the ion trap and beam-type experiments. This different behavior probably can be attributed to the shorter observation times and concomitant higher excitation energies in the in-source and QqTOF experiments compared to the long observation times and lower excitation energies relevant to the ion trap experiments. Based largely on elemental compositions derived from accurate mass measurements in QqTOF studies fragmentation schemes are proposed for the MH(+), [MH - NH(3)](+), and [MH - (HN = C(NH(2))(2))](+) ions.     

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.     

Characteristics of Ion Activation and Collision Induced Dissociation Using Digital Ion Trap Technology

Collision induced dissociation (CID) is one of the most established techniques for tandem mass spectrometry analysis. The CID of mass selected ion could be realized by ion resonance excitation with a digital rectangular waveform. The method is simple, and highly efficient CID result could be obtained by optimizing the experimental parameters, such as digital waveform voltage, frequency, and q value. In this work, the relationship between ion trapping waveform voltage and frequency at preselected q value, the relationship between waveform frequency and the q value at certain ion trapping voltage for optimum CID efficiency were investigated. Experiment results showed that the max CID efficiency of precursor reserpine ions can be obtained at different trapping waveform voltage and frequency when q and β are different. Based on systematic experimental analysis, the optimum experimental conditions for high CID efficiency can be calculated at any selected β or q. By using digital ion trap technology, the CID process and efficient fragmentation of parent ions can be realized by simply changing the trapping waveform amplitude, frequency, and the β values in the digital ion trap mass spectrometry. The technology and method are simple. It has potential use in ion trap mass spectrometry.

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Graphical Abstract ?

     

Determination of Cooling Rates in a Quadrupole Ion Trap

Collisional cooling rates of infrared excited ions are measured in a quadrupole ion trap (QIT) mass spectrometer at different combinations of temperature and pressure. Measurements are carried out by monitoring fragmentation efficiency of leucine enkephalin as a function of irradiation time by an infrared laser after a short excitation and incrementally increasing cooling periods. Cooling rates are observed to be directly related to bath gas pressure and inversely related to bath gas temperature. The cooling rate at typical ion trap operating pressure (1 mTorr) and temperature (room T) is faster than can be measured. At elevated temperature and the lowest pressure used for the studies, the rate of collisional cooling becomes negligible compared to the rate of radiative cooling.     

Axial CID and high pressure resonance CID in a miniature ion trap mass spectrometer using a discontinuous atmospheric pressure interface

Axial collision induced dissociation (CID) and high-pressure resonance CID were implemented and compared with normal low-pressure resonance CID in a miniature ion trap mass spectrometer to obtain more complete fragmentation spectra. Axial CID was realized simply by applying a potential to the discontinuous atmospheric pressure interface (DAPI) capillary without performing parent ion isolation before dissociation. High-pressure resonance CID employed a double-introduction pulse scan function, by means of which precursor ions isolated at low-pressure (<10⁻³ torr) were dissociated at high-pressure (0.1 torr-1 torr) with higher excitation energy, so that tandem MS of isolated precursor ions was achieved and extensive fragmentation was obtained. A simple peptide (Leu-enkephalin) and dye molecule (rhodamine B) ionized by ESI were used to investigate both methods and compare them with normal low-pressure resonance CID.     

Adaptation of a 3-D Quadrupole Ion Trap for Dipolar DC Collisional Activation

Means to allow for the application of a dipolar DC pulse to the end-cap electrodes of a three-dimensional (3-D) quadrupole ion trap for as short as a millisecond to as long as hundreds of milliseconds are described. The implementation of dipolar DC does not compromise the ability to apply AC waveforms to the end-cap electrodes at other times in the experiment. Dipolar DC provides a nonresonant means for ion acceleration by displacing ions from the center of the ion trap where they experience stronger rf electric fields, which increases the extent of micro-motion. The evolution of the product ion spectrum to higher generation products with time, as shown using protonated leucine enkephalin as a model protonated peptide, illustrates the broad-band nature of the activation. Dipolar DC activation is also shown to be effective as an ion heating approach in mimicking high amplitude short time excitation (HASTE)/pulsed Q dissociation (PQD) resonance excitation experiments that are intended to enhance the likelihood for observing low m/z products in ion trap tandem mass spectrometry.     

Fragmentation of oligosaccharide ions with 157 nm vacuum ultraviolet light

The 157 nm photofragmentation of native and derivatized oligosaccharides was studied in a linear ion trap and in a home-built matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometer, and the results were compared with collision-induced dissociation (CID) experiments. Photodissociation produces product ions corresponding to high-energy fragmentation pathways; for cation-derivatized oligosaccharides, it yields strong cross-ring fragment ions and provides better sequence coverage than low- and high-energy CID experiments. On the other hand, for native oligosaccharides, CID yielded somewhat better sequence coverage than photodissociation. The ion trap enables CID hybrid MS3 experiments on the high-energy fragment ions obtained from photodissociation.     

Dynamic collision-induced dissociation (DCID) in a quadrupole ion trap using a two-frequency excitation waveform: II. Effects of frequency spacing and scan rate

Dynamic CID of selected precursor ions is achieved by the application of a two-frequency excitation waveform to the end-cap electrodes during the mass instability scan of a quadrupole ion trap (QIT) mass spectrometer. This new method permits a shorter scanning time when compared with conventional on-resonance CID. When the excitation waveform consists of two closely-spaced frequencies, the relative phase-relationship of the two frequencies plays a critical role in the fragmentation dynamics. However, at wider frequency spacings (>10 kHz), these phase effects are diminished, while maintaining the efficacy of closely-spaced excitation frequencies. The fragmentation efficiencies and energetics of n-butylbenzene and tetra-alanine are studied under different experimental conditions and the results are compared at various scan rate parameters between 0.1 and 1.0 ms/Th. Although faster scan rates reduce the analysis time, the maximum observed fragmentation efficiencies rarely exceed 30%, compared with values in excess of 50% achieved at slower scan rates. The internal energies calculated from the simulations of n-butylbenzene at fast scan rates are approximately 4 eV for most experimental conditions, while at slow scan rates, internal energies above 5.5 eV are observed for a wide range of conditions. Extensive ITSIM simulations support the observation that slowing the scan rate has a similar effect on fragmentation as widening the frequency spacing between the two excitation frequencies. Both approaches generally enhance CID efficiencies and make fragmentation less dependent upon the relative phase angle between the excitation waveform and the ion motion.     

Nonlinear resonance effects during ion storage in a quadrupole ion trap

Contributions of higher-order fields to the quadrupolar storage field produce nonlinear resonances in the quadrupole ion trap. Storing ions with secular frequencies corresponding to these nonlinear resonances allows adsorption of power from the higher-order fields. This results in increased axial and radial amplitudes which can cause ion ejection and collision-induced dissociation (CID). Experiments employing long storage times and/or high ion populations, such as chemical ionization, ion-molecule reaction studies, and resonance excitation CID, can be particularly susceptible to nonlinear resonance effects. The effects of higher-order fields on stored ions are presented and the influence of instrumental parameters such as radiofrequency and direct current voltage (q_z and a_z values), ion population, and storage time are discussed.     

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.