Single-Frequency Ion Parking in a Digital 3D Quadrupole Ion Trap.

Single-frequency ion parking, a useful technique in electrospray mass spectrometry (ESI-MS), involves gas-phase charge-reduction ion/ion reactions in an electrodynamic ion trap in conjunction with the application of a supplementary oscillatory voltage to selectively inhibit the reaction rate of an ion of interest. The ion parking process provides a means for limiting the extent of charge reduction in a controlled fashion and allows for ions distributed over a range of charge states to be concentrated into fewer charge states (a single charge state under optimal conditions). As charge reduction inherently leads to an increase in the mass-to-charge (m/z) ratio of the ions, it is important that the means for storing and analyzing ions be able to accommodate ions of high m/z ratios. The so-called 'digital ion trap' (DIT), which uses a digital waveform as the trapping RF, has been demonstrated to be well-suited for the analysis of high m/z ions by taking advantage of its ability to manipulate the waveform frequency. In this study, the feasibility of ion parking in a 3D quadrupole ion trap operated as a DIT using a slow-amplitude single-frequency sine-wave for selective inhibition of an ion/ion reaction is demonstrated. A recently described model that describes ion parking has been adjusted for the DIT case and is used to interpret experimental data for proteins ranging in mass from 8600 Da to 467,000 Da.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions.

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high m/z ions. The additional tandem-MS (MSn) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at m/z > 50,000.

Digital ion trap mass analysis of high mass protein complexes using IR activation coupled with ion/ion reactions.

Native mass spectrometry (MS) focuses on measuring the masses of large biomolecular complexes and probing their structures. Large biomolecular complexes are readily introduced into mass spectrometers as gas-phase ions using electrospray ionization (ESI); however, the ions tend to be heavily adducted with solvent and salts, which leads to mass measurement errors. Various solution clean-up approaches can reduce the degree of adduction prior to introduction to the mass spectrometer. Gas-phase activation of trapped ions can provide additional adduct reduction, and charge reduction ion/ion reactions increase charge state separation. Together, gas-phase activation and charge reduction can combine to yield spectra of well separated charge states for improved mass measurements. A simple gas-phase collisional activation technique is to apply a dipolar DC (DDC) field to opposing electrodes in an ion trap. DDC activation loses its efficacy when ions are trapped at low q values, which is true of the high m/z ions generated by charge reduction ion/ion reactions. Digital ion trapping (DIT) readily traps high m/z ions at higher q values by varying trapping frequency rather than amplitude, but the low frequencies used to trap high m/z ions also decreases the efficacy of DDC activation. We demonstrate here using ions derived from GroEL that IR activation of ions shows no discrimination against high m/z ions trapped with DIT, because they can be focused equally well to the trap center to interact with the IR laser beam. Following pump out of excess background gas, IR activation can also induce efficient dissociation of the GroEL complex. This work demonstrates that IR activation is an effective approach for ion heating in native MS over the unusually wide range of charge states accessible via gas-phase ion/ion reactions.

Simultaneous Isolation of Nonadjacent m/z Ions Using Mirror Switching in an Electrostatic Linear Ion Trap.

Simultaneous isolation of ions of disparate mass-to-charge (m/z) ratios is demonstrated via appropriately timed pulsing of entrance and exit ion mirrors in an electrostatic linear ion trap (ELIT) mass spectrometer. Manipulation of the voltages of the entrance and exit mirrors, referred to as "mirror switching", has been demonstrated as a method in which ions can be both captured and isolated. High resolution isolation (>35 000) was previously demonstrated by selective gating of trapping electrodes to avoid ion lapping while closely spaced ions could continue to separate [ Johnson et al. Anal. Chem. 2019 , 91 , 8789 ]. In this work, we demonstrate that advantage can be taken of the ion lapping phenomenon in an ELIT to enable the simultaneous isolation of ions of disparate m/z ratios using mirror switching. This process is demonstrated with minimal ion loss using isotopologues of three carborane compounds ranging in m/z from 320 to 1020. Simultaneous isolation is demonstrated with the isolation of two and three peaks in separate isotopic distributions as well as with the isolation of alternating isotopologues within the same distribution. Such simultaneous isolation experiments are particularly useful when conducting experiments in which a mass calibrant is needed or when multiplexing in a tandem MS workflow.

Mirror Switching for High-Resolution Ion Isolation in an Electrostatic Linear Ion Trap.

Ion isolation was achieved via selective pulsing of the entrance and exit ion mirrors in an electrostatic linear ion trap mass spectrometer (ELIT). Mirror switching has been described previously as a method for capturing injected ions in ELIT devices. After ion trapping, mirror switching can be used as a method for ion isolation of successively narrower ranges of mass-to-charge (m/z) ratio. By taking advantage of the spatial separation of ions in an ELIT device, pulsing of the entrance and/or exit mirrors can release unwanted ions while continuing to store ions of interest. Furthermore, mirror switching can be repeated multiple times to isolate ions of very similar m/z values with minimal loss of the stored ions, as is demonstrated by the isolation of protonated l-glutamine and l-lysine (Δ m/z = 0.0364) from a mixture of the two amino acid ions and the isobaric mixture of [PC P-18:0/22:6] and [PC 19:0/19:0] (Δ m/z = 0.0575). As isolation is accomplished due to the spatial/temporal separation of ion packets within the ELIT, multiple reflection-time-of-flight (MR-TOF) mass spectra are shown to demonstrate separation in the ELIT at the time of isolation. An isolation resolution of greater than 35 000 fwhm is demonstrated here using a 5.25 in. ELIT. This resolution corresponds to the fwhm resolution necessary to reduce contaminant overlap of an equally abundant adjacent ion to 1% or less of the isolated ion intensity.

Triboluminescence from Pharmaceutical Formulations.

Triboluminescence (TL) is shown to enable selective detection of trace crystallinity within nominally amorphous solid dispersions (ASDs). ASDs are increasingly used for the preparation of pharmaceutical formulations, the physical stability of which can be negatively impacted by trace crystallinity introduced during manufacturing or storage. In the present study, TL measurements of a model ASD consisting of griseofulvin in polyethylene glycol produced limits of detection of 140 ppm. Separate studies of the particle size dependence of sucrose crystals and the dependence on polymorphism in clopidogrel bisulfate particles are both consistent with a mechanism for TL closely linked to the piezoelectric response of the crystalline fraction. Whereas disordered polymeric materials cannot support piezoelectric activity, molecular crystals produced from homochiral molecules adopt crystal structures that are overwhelmingly symmetry-allowed for piezoelectricity. Consequently, TL may provide a broadly applicable and simple experimental route for sensitive detection of trace crystallinity within nominally amorphous materials.

Increasing the Upper Mass/Charge Limit of a Quadrupole Ion Trap for Ion/Ion Reaction Product Analysis via Waveform Switching.

Quadrupole ion traps (QITs) are versatile platforms for performing experiments with gas-phase ions due to their abilities to store ions of both polarities and to conduct MSn experiments. The QIT is particularly useful as a reaction cell for ion/ion reactions. In the case of an ion/ion reaction experiment in a QIT, multiply charged reactant ions may initially be of relatively low m/z (e.g., m/z < 1000) whereas the product ions can be one or more orders of magnitude higher in m/z (e.g., m/z > 100,000). Several factors can limit the m/z range over which an ion/ion reaction experiment can be conducted. These include (1) the efficiency of the detector, (2) the m/z range over which oppositely charged ions can be mutually stored, and (3) the m/z range over which ions can be mass selectively ejected into an external detector. High-frequency waveforms provide larger m/z trapping ranges for mutual storage of oppositely charged ions whereas low-frequency waveforms provide better trapping for very high m/z product ions. Presented here is a method that switches from a high-frequency sine wave prior to and during an ion/ion reaction to a low-frequency square wave to eject low m/z reagent ions and improves confinement of the product ions before mass-selective ejection by scanning the frequency of the square wave. This approach addresses the third issue, which is the primary limiting factor with QITs operated at high RF (e.g., > 900 MHz). Graphical Abstract.