Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer

The use of a Q-q-Q(linear ion trap) instrument to obtain product ion spectra is described. The instrument is based on the ion path of a triple quadrupole mass spectrometer with Q3 operable as either a conventional RF/DC quadrupole mass filter or a linear ion trap mass spectrometer with axial ion ejection. This unique ion optical arrangement allows de-coupling of precursor ion isolation and fragmentation from the ion trap itself. The result is a high sensitivity tandem mass spectrometer with triple quadrupole fragmentation patterns and no inherent low mass cut-off. The use of the entrance RF-only section of the instrument as accumulation ion trap while the linear ion trap mass spectrometer is scanning enhances duty cycles and results in increased sensitivities by as much as a factor of 20. The instrument is also capable of all of the triple quadrupole scans including multiple-reaction monitoring (MRM) as well as precursor and constant neutral loss scanning. The high product ion scanning sensitivity allows the recording of useful product ion spectra near the MRM limit of quantitation.     

Electrospray ionization-Fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity

The dynamic range of Fourier transform ion cyclotron mass spectrometry (FTICR) is typically limited by the useful charge capacity of an FTICR cell (to approximately 10(6) to 10(7) elementary charges) and the minimum number of ions required to produce a useful signal (approximately 10(2) elementary charges). We show that the expansion of the dynamic range by 2 orders of magnitude can be achieved by preselecting lower abundance species in a quadrupole interface to an electrospray ionization (ESI) source. Ion preselection is then followed by ion accumulation in external to the FTICR cell a linear (2-D) quadrupole trap and subsequent transfer to the region of high magnetic field for gated trapping in the FTICR cell. Two modes of ion preselection, using either the quadrupole filtering mode or rf-only dipolar excitation, were studied and mass resolutions of 30 to 100 were achieved for selective external ion accumulation of peptides and proteins with molecular weights ranging from 500 to 17,000 Da. The ability to selectively eject the most abundant species before trapping in the FTICR has enormous practical benefits for increasing the sensitivity and dynamic range of measurements.     

A new technique for unbiased external ion accumulation in a quadrupole two-dimensional ion trap for electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

External ion accumulation in a two-dimensional (2D) multipole trap has been shown to increase the sensitivity, dynamic range and duty cycle of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. However, it is important that trapped ions be detected without significant bias at longer accumulation times in the external 2D multipole trap. With increasing ion accumulation time pronounced m/z discrimination was observed when trapping ions in an accumulation quadrupole. In this work we show that superimposing lower rf-amplitude dipolar excitation over the main rf-field in the accumulation quadrupole results in disruption of the m/z discrimination and can potentially be used to achieve unbiased external ion accumulation with FTICR.     

Restrained ion population transfer: a novel ion transfer method for mass spectrometry

Fourier transform ion cyclotron resonance (FTICR) mass spectrometers function such that the ion accumulation event takes place in a region of higher pressure outside the magnetic field which allows ions to be thermally cooled before being accelerated toward the ICR cell where they are decelerated and re-trapped. This transfer process suffers from mass discrimination due to time-of-flight effects. Also, trapping ions with substantial axial kinetic energy can decrease the performance of the FTICR instrument compared with the analysis of thermally cooled ions located at the trap center. Therefore, it is desirable to limit the energy imparted to the ions which results in lower applied trap plate potentials and reduces the spread in axial kinetic energy. The approach presented here for ion transfer, called restrained ion population transfer or RIPT, is designed to provide complete axial and radial containment of an ion population throughout the entire transfer process from the accumulation region to the ICR cell, eliminating mass discrimination associated with time-of-flight separation. This was accomplished by use of a number of quadrupole segments arranged in series with independent control of the direct current (DC) bias voltage applied to each segment of the quadrupole ion guide. The DC bias voltage is applied in such a way as to minimize the energy imparted to the ions allowing transfer of ions with low kinetic energy from the ion accumulation region to the ICR cell. Initial FTICR mass spectral data are presented that illustrate the feasibility of RIPT. A larger m/z range for a mixture of peptides is demonstrated compared with gated trapping. The increase in ion transfer time (3 ms to 130 ms) resulted in an approximately 11% decrease in the duty cycle; however this can be improved by simultaneously transferring multiple ion populations with RIPT. The technique was also modeled with SIMION 7.0 and simulation results that support our feasibility studies of the ion transfer process are presented.     

A new hybrid electrospray Fourier transform mass spectrometer: design and performance characteristics

A new hybrid electrospray quadrupole Fourier transform mass spectrometry (FTMS) instrument design is shown and characterized. This instrument involves coupling an electrospray source and mass-resolving quadrupole, ion accumulation, and collision cell linear ion trap system developed by MDS Sciex with a home-built ion guide and ion cyclotron resonance (ICR) cell. The iterative progression of this design is shown. The final design involves a set of hexapole ion guides to transfer the ions from the accumulation/collision trap through the magnetic field gradient and into the cell. These hexapole ion guides are separated by a thin gate valve and two conduction limits to maintain the required <10(-9) mbar vacuum for FTICR. Low-attomole detection limits for a pure peptide are shown, 220 000 resolving power in broadband mode and 820 000 resolving power in narrow-band mode are demonstrated, and mass accuracy in the <2 ppm range is routinely available provided the signal is abundant, cleanly resolved, and internally calibrated. This instrument design provides high experimental flexibility, allowing Q2 CAD, SORI-CAD, IRMPD, and ECD experiments with selected ion accumulation as well as experiments such as nozzle skimmer dissociation. Initial top-down mass spectrometry experiments on a protein is shown using ECD.     

Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry

Undesired fragmentation of electrospray generated ions in an rf multipole traps can be problematic in many applications. Of special interest here is ion dissociation in a 2-D quadrupole ion trap external to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) used in proteomic studies. In this work, we identified the experimental parameters that determine the efficiency of ion fragmentation. We have found that under the pressure conditions used in this study there is a specific combination of the radial and axial potential well depths that determines the fragmentation threshold. This combination of rf and dc fields appears to be universal for ions of different mass-to-charge ratios, molecular weights, and charge states. Such universality allows the fragmentation efficiency of the trapped ions in the course of capillary liquid chromatography (LC) separation studied to be controlled and can increase the useful duty cycle and dynamic range of a FTICR mass spectrometer equipped with an external rf only 2-D quadrupole ion trap.     

Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer

Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolutions of 60 000 were achieved with mass accuracies averaging below 1.4 ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS.     

Gas-phase ion chemistry and organic chemistry-the story of a hybrid six sector mass spectrometer--the "AutoSpec 6F"

The AutoSpec 6F mass spectrometer is a large, floor standing instrument comprising a pair of commercial EBE geometry (AutoSpec) mass spectrometers coupled in series to provide an hybrid EBE-EBE configuration, (E and B being respectively electrostatic and magnetic sectors.) It was designed in close collaboration between Professor R. Flammang and VG Analytical in Manchester, UK. It was equipped with five collision cells and allowed the recording of high energy CID (collision induced dissociation), MIKES (mass analyzed ion kinetic energy spectrometry) and NRMS (neutralization re-ionization mass spectrometry) data as well as consecutive MSn analyses. The field-free regions between sectors allowed the study of unimolecular decomposition products from long-lived metastable ions. The mass spectrometer became even more versatile when an RF-only quadrupole collision cell was installed between the second and the third electric sector. This allowed the study of associative ion/molecule reactions in the low kinetic energy regime. Bimolecular chemical reactions were performed inside the quadrupole cell when a neutral reagent was introduced and the reaction products were analyzed by high energy CID in the downstream sectors. This paper tells the history and summarizes the capabilities of this versatile instrument.     

External accumulation of ions for enhanced electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.     

Analytical atomic spectroscopy using ion trap devices

Investigations using ion trap devices for analytical atomic spectroscopy purposes have focused on the use of an inductively coupled plasma (ICP) ion source with ion trap mass spectrometric (ITMS) detection. Initial studies were conducted with an instrument assembled by simply appending an ion trap as the detector to a fairly conventional ICP/MS instrument, i.e. leaving an intermediate linear quadrupole between the plasma source and the ion trap. The principal advantages found with this system include the destruction of nearly all problematic and typical ICP/MS polyatomic ions (e.g., ArH(+), ArO(+), ArCl(+), Ar(2)(+), etc) and a dramatic reduction of the primary plasma source ion, Ar(+). These results prompted the development of a second-generation plasma source ion trap instrument in which direct coupling of the ICP and ion trap has been effected (i.e. no intermediate linear quadrupole); the same performance benefits have been largely preserved. Initial operation of this instrument is described, characterized, and compared to the originally described ICP/ITMS and conventional ICP/MS systems. In addition, experiments aimed at improving ICP/ITMS sensitivity and selectivity using broadband resonance excitation techniques are described. Finally, the potential for laser optical detection of trapped ions for analytical purposes is speculated upon.     

Comparison of Functional Group Selective Ion–Molecule Reactions of Trimethyl Borate in Different Ion Trap Mass Spectrometers

We report here a comparison of the use of diagnostic ion–molecule reactions for the identification of oxygen-containing functional groups in Fourier-transform ion cyclotron resonance (FTICR) and linear quadrupole ion trap (LQIT) mass spectrometers. The ultimate goal of this research is to be able to identify functionalities in previously unknown analytes by using many different types of mass spectrometers. Previous work has focused on the reactions of various boron reagents with protonated oxygen-containing analytes in FTICR mass spectrometers. By using a LQIT modified to allow the introduction of neutral reagents into the helium buffer gas, this methodology has been successfully implemented to this type of an ion trap instrument. The products obtained from the reactions of trimethyl borate (TMB) with various protonated analytes are compared for the two instruments. Finally, the ability to integrate these reactions into LC-MS experiments on the LQIT is demonstrated.     

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.     

Transmission mode ion/ion proton transfer reactions in a linear ion trap

A new method is described for effecting ion/ion proton transfer reactions that involves storage of analyte ions while oppositely charged ions are transmitted through the stored ion population. In this approach, the products are captured and stored in the linear ion trap for subsequent mass analysis. Charge reduction of multiply charged protein ions is used as an example to illustrate the analytical usefulness of this method. In another variation of the transmission mode ion/ion reaction approach, two charge inversion experiments, implemented by passing analyte ions through a population of multiply charged reagent ions in a LIT, are also demonstrated. A pulsed dual ion source approach coupled with a hybrid triple quadrupole/linear ion trap instrument was used to demonstrate these two methods. The results for ion/ion reactions implemented using these so-called "transmission mode" experiments were comparable to those acquired using the more conventional mutual storage mode, both in terms of efficiency and information content of the spectra. An advantage of transmission mode experiments compared with mutual storage mode experiments is that they do not require any specialized measures to be taken to enable the simultaneous storage of oppositely charged ions.     

Analytical atomic spectroscopy using ion trap devices

Investigations using ion trap devices for analytical atomic spectroscopy purposes have focused on the use of an inductively coupled plasma (ICP) ion source with ion trap mass spectrometric (ITMS) detection. Initial studies were conducted with an instrument assembled by simply appending an ion trap as the detector to a fairly conventional ICP/MS instrument, i.e. leaving an intermediate linear quadrupole between the plasma source and the ion trap. The principal advantages found with this system include the destruction of nearly all problematic and typical ICP/MS polyatomic ions (e.g., ArH⁺, ArO⁺, ArCl⁺, Ar₂⁺, etc) and a dramatic reduction of the primary plasma source ion, Ar⁺. These results prompted the development of a second-generation plasma source ion trap instrument in which direct coupling of the ICP and ion trap has been effected (i.e. no intermediate linear quadrupole); the same performance benefits have been largely preserved. Initial operation of this instrument is described, characterized, and compared to the originally described ICP/ITMS and conventional ICP/MS systems. In addition, experiments aimed at improving ICP/ITMS sensitivity and selectivity using broadband resonance excitation techniques are described. Finally, the potential for laser optical detection of trapped ions for analytical purposes is speculated upon.     

Transmission mode ion/ion reactions in the radiofrequency‐only ion guide of hybrid tandem mass spectrometers

Transmission mode ion/ion reactions have been performed within the first quadrupole, the Q0 radiofrequency (RF)‐only quadrupole, of two types of hybrid tandem mass spectrometers (viz., triple quadrupole/linear ion trap and QqTOF instruments). These transmission mode reactions involved the storage of either the reagent species and the transmission of the analyte species through the Q0 quadrupole for charge inversion reactions or the storage of the analyte ions and transmission of the reagent ions as in charge reduction experiments. A key advantage to the use of transmission mode ion/ion reactions is that they do not require any instrument hardware modifications to provide interactions of oppositely charged ions and can be implemented in any instrument that contains a quadrupole or linear ion trap. The focus of this work was to investigate the potential of using the RF‐only quadrupole ion guide positioned prior to the first mass‐resolving element in a tandem mass spectrometer for ion/ion reactions. Two types of exemplary experiments have been demonstrated. One involved a charge inversion reaction and the other involved a charge reduction reaction in conjunction with ion parking. Ion/ion reactions proved to be readily implemented in Q0 thereby adding significantly greater experimental flexibility in the use of ion/ion reaction experiments with hybrid tandem mass spectrometers. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.     

Development and characterization of an electrospray ionization ion trap/linear time-of-flight mass spectrometer

On-line analysis of compounds from solution has been greatly facilitated by the advent of electrospray ionization mass spectrometry (ESI-MS). Although quadrupole mass analyzers are most commonly used with ESI at present, time-of-flight (TOF) mass spectrometers offer several potential advantages including high data acquisition rates, which are desirable for fast separation techniques. One method of coupling ESI and TOF uses an ion trap for temporary storage and accumulation of the electrosprayed ions prior to TOF mass analysis. Previous studies have not fully addressed the effects of several key variables on the analytical capabilities of this type of instrument. In this study, the characterization of an ion trap/linear TOF instrument for ESI is described. The behavior of various analytes is divided into two separate groups; each one is found to have its own optimal set of operating conditions. The reasons for the observed differences between groups are explored. Issues relevant to mass resolution, sensitivity, mass range, mass-to-charge ratio discrimination, and mass measurement accuracy are addressed. Finally, it is suggested that the analytical capability of this type of instrument could be significantly improved by changing the ion optics from the existing focusing lenses to a rf-only quadrupole lens.     

Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules

Recently, linear ion traps (LITs) have been combined with quadrupole (Q), time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). LITs can be used either as ion accumulation devices or as commercially available, stand-alone mass spectrometers with MSn capabilities. The combination of triple quadrupole MS with LIT technology in the form of an instrument of configuration QqLIT, using axial ejection, is particularly interesting, because this instrument retains the classical triple quadrupole scan functions such as selected reaction monitoring (SRM), product ion (PI), neutral loss (NL) and precursor ion (PC) while also providing access to sensitive ion trap experiments. For small molecules, quantitative and qualitative analysis can be performed using the same instrument. In addition, for peptide analysis, the enhanced multiply charged (EMC) scan allows an increase in selectivity, while the time-delayed fragmentation (TDF) scan provides additional structural information. Various methods of operating the hybrid instrument are described for the case of the commercial Q TRAP (AB/MDS Sciex) and applications to drug metabolism analysis, quantitative confirmatory analysis, peptides analysis and automated nanoelectrospray (ESI-chip-MS) analysis are discussed.     

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry

A primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis.     

Electrospray ionization—Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: Instrument design and initial results

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10⁶ has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.