Transmission mode ion/ion electron-transfer dissociation in a linear ion trap

Two related methods for effecting electron-transfer dissociation (ETD) are described that involve either the storage of analyte cations in a linear ion trap while reagent anions are transmitted through the cations or storage of the reagent anions with transmission of the analyte cations. In the former approach, the ETD products are captured and stored in the linear ion trap for subsequent mass analysis. In the latter approach, the ETD products pass through the linear ion trap and must be collected or directly mass-analyzed by an external device. In the present study, another linear ion trap is placed in series with the ion trap where the ion/ion reaction was employed. A pulsed dual ion source approach coupled with a hybrid triple quadrupole/linear ion trap instrument was used to illustrate these methods. The two approaches give similar results in terms of the identities and relative abundances of the ETD products. Under optimum conditions, the two approaches also give comparable extents of ion/ion reactions for the same reaction time. Also, conversions of precursor ions to product ions over the same reaction time are similar to those noted for experiments in which ions of both polarities are stored simultaneously. These approaches, therefore, provide expanded experimental options for the use of ETD. An advantage of transmission mode experiments that they hold over mutual storage mode experiments is that they do not require that any specialized measures be taken to enable the simultaneous storage of oppositely charged ions.     

Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

Bidirectional ion transfer between quadrupole arrays: MSn ion/ion reaction experiments on a quadrupole/time-of-flight tandem mass spectrometer

Methods for bidirectional ion transmission between distinct quadrupole arrays were developed on a quadrupole/time-of-flight tandem mass spectrometer (QqTOF) containing three quadrupoles (ion guide Q0, mass filter Q1, and collision cell Q2) and a reflectron TOF analyzer, for the purpose of implementing multistage ion/ion reaction experiments. The transfer efficiency, defined as the percentage of ions detected after two transfer steps relative to the initial ion abundance, was found to be about 60% between Q2 and Q0 (with passage through the intermediate array (Q1)) and almost 100% between Q2 and Q1. Efficient ion transfer enabled new means for executing MSn experiments on an instrument of this type by operating Q1 in rf/dc mode for performing multiple steps of precursor/product ion isolation while passing ions through Q1 or trapping ions in Q1. In the latter case, the Q1 functioned as a linear ion trap. Either collision induced dissociation (CID) or ion/ion reactions can be conducted in between each stage of mass analysis. MS3 or MS4 experiments were developed to illustrate the charge increase of peptide ions via two steps of charge inversion ion/ion reactions, CID of electron-transfer dissociation (ETD) products and CID of a metal-peptide complex formed from ion/ion reactions.     

Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions

Cationic peptide electron-transfer products that do not fragment spontaneously are exposed to ion trap collisional activation immediately upon formation while they pass through a high-pressure collision cell (Q2), where the electron-transfer reagent anions are stored. Radial ion acceleration, which is normal to the ion flow, is implemented by applying an auxiliary dipolar alternating current to a pair of opposing rods of the Q2 quadrupole array at a frequency in resonance with the surviving electron-transfer products. Collisional cooling of cations in the pressurized Q2 ensures efficient overlap of the positive and negative ions for ion/ion reactions and also gives rise to relatively long residence times (milliseconds) for ions in Q2, making it possible to fragment ions via radial excitation during their axial transmission. The radial activation for transmission mode electron-transfer ion/ion reactions has been demonstrated with a doubly protonated tryptic peptide, a triply protonated phosphopeptide, and [M + 7H]7+ ions of ubiquitin. In all cases, significant increases in fragment ion yields and structural information from electron-transfer dissociation (ETD) were observed, suggesting the utility of this method for improving transmission mode ETD performance for relatively low charge states of peptides and proteins.     

Oligonucleotide mixture analysis via electrospray and ion/ion reactions in a quadrupole ion trap

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz., positive oligonucleotides/negative charge-transfer agent versus negative oligonucleotides/positive charge-transfer agent. Anions derived from perfluorocarbons appear to be directly applicable to mixtures of positive ions derived from electrospray of oligonucleotides, in direct analogy with positive protein ions. Conditions for forming positive oligonucleotide ions devoid of adducts were more difficult to establish than for forming relatively clean negative oligonucleotide ions. A new approach for manipulating negative ion charge states in the ion trap is described and is based on use of the electric field of the positive charge-transfer agent for storage of high-mass negative ions formed during the ion/ion reaction period. Oxygen cations are shown to be acceptable for charge-state manipulation of mixed-base oligomers but induce fragmentation in polyadenylate homopolymers. Protonated isobutylene (C4H9+), on the other hand, is shown to induce significantly less fragmentation of polyadenylate homopolymers.     

High-pressure ion source combined with an in-axis ion trap mass spectrometer. 2. Application of selective low-pressure negative ion chemical ionization

Negative ion chemical ionization was carried out using a quadrupole ion trap mass spectrometer with selected reactant negative ions, primarily injected from a homemade dual EI/CI external ion source. Hence, selective ion/molecule reactions were provided according to the reaction time, which induce a greater control over bimolecular ionization mechanisms than in conventional a high-pressure ion source combined with beam instruments, where several competitive ionization processes take place mainly due to source conditions (e.g., temperature, pressure, and repeller). By selecting the reactant ions, ion/molecule reactions were specifically produced (i.e., charge exchange, proton transfer, nucleophilic substitution, and/or alpha-beta elimination) with several organic target compounds. Gas-phase reactivity of phosphorus- and nitrogen-containing compounds (such as phosphonates as representative for chemical warfare agents and phosphorothionates, phosphorodithionates, and triazines for pesticides) as well as dinitro aromatic compounds (for pesticides) has been explored, in the present work, to ensure further unambiguous detection.     

Continuous real-time analysis of products from the reaction of some monoterpenes with ozone using atmospheric sampling glow discharge ionization coupled to a quadrupole ion trap mass spectrometer

An on-line technique has been demonstrated for the analysis of photochemical oxidation reaction products. The technique is based on the direct introduction of gas and particulate oxidation products into a custom-built atmospheric sampling glow discharge ionization source (ASGDI) coupled to a quadrupole ion trap mass spectrometer (QITMS). Operational parameters of the ASGDI system were investigated to determine their influence on the ion signal for the analysis of oxidation products in real time. These parameters include the discharge current, ion accumulation time, and type of reagent gas. Reference mass spectra from standards were generated for a variety of biogenic compounds and terpene reaction products containing keto, hydroxy, aldehyde, carboxylic acid, or epoxy groups to better understand the fragmentation that occurs in the glow discharge ion source. Results are presented for ozonolysis reactions of four biogenic monoterpenes (alpha-pinene, beta-pinene, D-limonene, Delta(3)-carene) monitored with the ASGDI quadrupole ion trap to demonstrate the ability to obtain real-time measurements. The reaction products identified with ASGDI-QITMS correspond to those products identified with other techniques, including on-line atmospheric pressure chemical ionization techniques. Efficient differentiation of multifunctional products including mono-/di-/hydroxy-/keto-carboxylic acid and keto-/hydroxy-aldehyde was possible by use of the MS/MS capability of the instrument.     

A tandem ion trap/ion mobility spectrometer

A tandem quadrupole ion trap/ion mobility spectrometer (QIT/IMS) has been constructed for structural analysis based on the gas-phase mobilities of mass-selected ions. The instrument combines the ion accumulation, manipulation, and mass-selection capabilities of a modified ion trap mass spectrometer with gas-phase electrophoretic separation in a custom-built ion mobility drift cell. The quadrupole ion trap may be operated as a conventional mass spectrometer, with ion detection using an off-axis dynode/multiplier arrangement, or as an ion source for the IMS drift cell. In the latter case, pulses of ions are ejected from the trap and transferred to the drift cell where mobility in the presence of helium buffer gas is determined by the collision cross section of the ion. Ions traversing the drift cell are detected by an in-line electron multiplier and the data processed with a multichannel scaler. Preliminary data are presented on instrumental performance characteristics and the application of QIT/ IMS to structural and conformational studies of aromatic ions and protonated amine/crown ether noncovalent complexes generated via ion/molecule reactions in the ion trap.     

Parallel ion parking: improving conversion of parents to first-generation products in electron transfer dissociation

Electron-transfer dissociation (ETD) in a tandem mass spectrometer is an analytically useful ion/ion reaction technique for deriving polypeptide sequence information, but its utility can be limited by sequential reactions of the products. Sequential reactions lead to neutralization of some products, as well as to signals from products derived from multiple cleavages that can be difficult to interpret. A method of inhibiting sequential ETD fragmentation in a quadrupole ion trap is demonstrated here for the reaction of a triply protonated peptide with nitrobenzene anions. A tailored waveform (in this case, a filtered noise field) is applied during the ion/ion reaction time to accelerate simultaneously first-generation product ions and thereby inhibit their further reaction. This results in a approximately 50% gain in the relative yield of first-generation products and allows for the conversion of more than 90% of the original parent ions into first-generation products. Gains are expected to be even larger when higher charge-state cations are used, as the rates of sequential reaction become closer to the initial reaction rate.     

High-pressure ion source combined with an in-axis ion trap mass spectrometer. 1. Instrumentation and applications

A new combination of a dual EI/CI ion source with a quadrupole ion trap mass spectrometer has been realized in order to efficiently produce negative ions in the reaction cell. Analysis of volatile compounds was performed under negative ion chemical ionization (NICI) during a reaction period where selected reactant negative ions, previously produced in the external ion source, were allowed to interact with molecules, introduced by hyphenated techniques such as gas chromatography. The O2*-, CH3O-, and Cl- reactant ions were used in this study to ensure specific ion/molecule interactions such as proton transfer, nucleophilic displacement, or charge exchange processes, respectively leading to even-electron species, i.e., deprotonated [M - H]- molecules, diagnostic [M - R]- ions, or odd-electron M*- molecular species. The reaction orientation depends on the thermochemistry of reactions within kinetic controls. First analytical results are presented here for the trace-level detection of several contaminants under NICI/Cl- conditions. Phosphorus-containing compounds (malathion, ethyl parathion, and methyl parathion as representative for pesticides) and nitro-containing compounds (2,4,6-trinitrotoluene for explosive material) have been chosen in order to explore the analytical ability of this promising instrumental coupling.     

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.     

An interface with a linear quadrupole ion guide for an electrospray-ion trap mass spectrometer system

A new ion sampling interface for an electrospray ionization 3D ion trap mass spectrometer system is described. The interface uses linear rf quadrupoles as ion guides and ion traps to enhance the performance of the 3D trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. A resolution of ejection of up to 254 is demonstrated for protonated reserpine ions (m/z 609.3). A composite waveform with a notch in frequency space is used to eject a wide range of matrix ions and to isolate trace analyte ions in a linear quadrupole before ions are injected into the 3D trap. This is useful to overcome space charge problems in the 3D trap caused by excess matrix ions. For trace reserpine in a 500-fold molar excess of poly(propylene glycol) (PPG), it is demonstrated that the resolution and sensitivity of the 3D trap can be increased dramatically with ejection of the excess PPG matrix ions. In comparison to ejection of matrix ions in the 3D trap with a similar broad-band waveform, a 5-fold increase in sensitivity with a 7 times shorter acquisition time was achieved.     

Development of a proton-transfer reaction-linear ion trap mass spectrometer for quantitative determination of volatile organic compounds

Currently, proton-transfer reaction mass spectrometry (PTR-MS) allows for quantitative determination of volatile organic compounds in real time at concentrations in the low ppt range, but cannot differentiate isomers or isobaric molecules, using the conventional quadrupole mass filter. Here we pursue the application of linear quadrupole ion trap (LIT) mass spectrometry in combination with proton-transfer reaction chemical ionization to provide the advantages of specificity from MS/MS. A commercial PTR-MS platform composed of a quadrupole mass filter with the addition of end cap electrodes enabled the mass filter to operate as a linear ion trap. The rf drive electronics were adapted to enable the application of dipolar excitation to opposing rods, for collision-induced dissociation (CID) of trapped ions. This adaptation enabled ion isolation, ion activation, and mass analysis. The utility of the PTR-LIT was demonstrated by distinguishing between the isomeric isoprene oxidation pair, methyl vinyl ketone (MVK) and methacrolein (MACR). The CID voltage was adjusted to maximize the m/ z 41 to 43 fragment ratio of MACR while still maintaining adequate sensitivity. Linear calibration curves for MVK and MACR fragments at m/ z 41 and 43 were obtained with limits of detection of approximately 100 ppt, which should enable ambient measurements. Finally, the PTR-LIT method was compared to an established GC/MS method by quantifying MVK and MACR production during a smog chamber isoprene-NO x irradiation experiment.     

Rapidly alternating transmission mode electron-transfer dissociation and collisional activation for the characterization of polypeptide ions

Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type collisional activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.     

Charge determination of product ions formed from collision-induced dissociation of multiply protonated molecules via ion/molecule reactions.

The use of ion/molecule reactions involving multiply protonated ions derived from electrospray for the determination of the charges of product ions formed from collision-induced dissociation is described. The experiments are carried out with a quadrupole ion trap capable of multiple stages of mass spectrometry. The approach is illustrated with proton transfer from a product ion from quadruply protonated melittin, and from a product ion from the (M + 20H)20+ ion from horse myoglobin, to 1,6-diaminohexane. The major product ion from quadruply protonated bovine insulin is used to illustrate the use of a clustering reaction with 1,6-diaminohexane. The ion trap is shown to be a particularly useful tool for employing both collisional activation and low-energy ion/molecule reactions in the same experiment to determine product ion charge.     

Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition

Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.     

Identification of anomeric configuration of underivatized reducing glucopyranosyl-glucose disaccharides by tandem mass spectrometry and multivariate analysis

The possibility of discrimination of the anomeric configuration (alpha or beta) of underivatized reducing glucopyranosyl-glucose disaccharides, using a hybrid mass spectrometer Q-TOF 2 (Micromass), a linear ion trap LXQ (Thermo), and a triple quadrupole Quattro (Micromass) with an electrospray source (ESI) was investigated. Differences observed in the relative abundances of specific product ions obtained from collisionally induced dissociation of the [M + Li]+ adducts were statistically analyzed, and discriminant analysis was performed. MANOVA has shown that anomeric configuration has influence on the combined dependent variables (relative abundances of m/z product ions) in all the three mass spectrometers used (Q-TOF 2, LIT, and QqQ). Discriminant analysis has shown that, in all instruments, it is possible to discriminate anomeric configurations and to build a diagnostic model. These diagnostic differences are even more relevant considering that no derivatization procedures are needed for obtaining this structural information. The Q-TOF 2 instrument has been shown to give data that allowed us to build a model with better discriminant power (Wilks' lambda value of 0.014) followed by the QqQ instrument (Wilks' lambda value of 0.029) and the LIT instrument (Wilks' lambda value of 0.037).     

Mass analysis of mobility-selected ion populations using dual gate, ion mobility, quadrupole ion trap mass spectrometry

An electrospray ionization, dual gate, ion mobility, quadrupole ion trap mass spectrometer (ESI-DG-IM-QIT-MS) was constructed and evaluated for its ability to select mobility-filtered ions prior to mass analysis. While modification of the common signal-averaged ion mobility experiment was required, no modifications to the QIT were necessary. The dual gate scanning mode of operation was used to acquire mobility spectra, whereas the single mobility monitoring experiment selectively filtered ions for concentration and subsequent fragmentation within the QIT. Ion mobility separation of positively charged peptides and negatively charged carbohydrates, followed by MS fragmentation, was demonstrated. For a 1-min acquisition time, it was possible to obtain complete de novo sequence information for the examined peptides. Fragmentation of the negative carbohydrate chlorine adducts yielded ions characteristic of cross-ring and glycosidic bond cleavage. Previous unions of atmospheric pressure ion mobility and mass spectrometry have been limited in their ability to reproducibly obtain MSn data for mobility separation ions. The union of high-pressure ion mobility with quadrupole ion trap mass spectrometry presents the unique opportunity to obtain more detailed information regarding the chemistries of gas-phase ions.     

Ion trap versus low-energy beam-type collision-induced dissociation of protonated ubiquitin ions

The beam-type and ion trap collision-induced dissociation (CID) behaviors of protonated bovine ubiquitin ions were studied for charge states ranging from +6 to +12 on a modified triple quadrupole/linear ion trap tandem mass spectrometer. Both beam-type CID and ion trap CID were conducted in a high-pressure linear ion trap, followed by proton-transfer ion/ion reactions to reduce the charge states of product ions mostly to +1. The product ions observed under each activation condition were predominantly b- and y-type ions. Fragmentation patterns showed a much stronger dependence on parent ion charge state with ion trap CID than with beam-type CID using nitrogen as the collision gas, with preferential cleavages C-terminal to aspartic acid at relatively low charge states, nonspecific fragmentation at moderate charge states, and favored cleavages N-terminal to proline residues at high charge states. In the beam-type CID case, extensive cleavage along the protein backbone was noted, which yielded richer sequence information (77% of backbone amide bond cleavages) than did ion trap CID (52% of backbone amide bond cleavages). Collision gas identity and collision energy were also evaluated in terms of their effects on the beam-type CID spectrum. The use of helium as collision gas, as opposed to nitrogen, resulted in CID behavior that was sensitive to changes in collision energy. At low collision energies, the beam-type CID data resembled the ion trap CID data with preferential cleavages predominant, while at high collision energies, nonspecific fragmentation was observed with increased contributions from sequential fragmentation.