Ion parking in native mass spectrometry.

A forced, damped harmonic oscillator model for gas-phase ion parking using single-frequency resonance excitation is described and applied to high-mass ions of relevance to native mass spectrometry. Experimental data are provided to illustrate key findings revealed by the modelling. These include: (i) ion secular frequency spacings between adjacent charge states of a given protein are essentially constant and decrease with the mass of the protein (ii) the mechanism for ion parking of high mass ions is the separation of the ion clouds of the oppositely-charged ions with much less influence from an increase in the relative ion velocity due to resonance excitation, (iii) the size of the parked ion cloud ultimately limits ion parking at high m/z ratio, and (iv) the extent of ion parking of off-target ions is highly sensitive to the bath gas pressure in the ion trap. The model is applied to ions of 17 kDa, 467 kDa, and 2 MDa while experimental data are also provided for ions of horse skeletal muscle myoglobin (≈17 kDa) and ß-galactosidase (≈467 kDa). The model predicts and data show that it is possible to effect ion parking on a 17 kDa protein to the 1+ charge state under trapping conditions that are readily accessible with commercially available ion traps. It is also possible to park ß-galactosidase efficiently to a roughly equivalent m/z ratio (i.e., the 26+ charge state) under the same trapping conditions. However, as charge states decrease, analyte ion cloud sizes become too large to allow for efficient ion trapping. The model allows for a semi-quantitative prediction of ion trapping performance as a function of ion trapping, resonance excitation, and pressure conditions.

Adaptation and Operation of a Quadrupole/Time-of-Flight Tandem Mass Spectrometer for High Mass Ion/Ion Reaction Studies.

A commercial quadrupole/time-of-flight tandem mass spectrometer has been modified and evaluated for its performance in conducting ion/ion reaction studies involving high mass (>100 kDa) ions. Modifications include enabling the application of dipolar AC waveforms to opposing rods in three quadrupole arrays in the ion path. This modification allows for resonance excitation of ions to effect ion activation, selective ion isolation, and ion parking. The other set of opposing rods in each array is enabled for the application of dipolar DC voltages for the purpose of broad-band (non-selective) ion heating. The plates between each quadrupole array are enabled for the application of either DC or AC (or both) voltages. The use of AC voltages allows for the simultaneous storage of ions of opposite polarity, thereby enabling mutual storage ion/ion reactions. Ions derived from nano-electrospray ionization of GroEL and ß-galactosidase under native conditions were used to evaluate limits of instrument performance, in terms of m/z range, ion isolation, and ion storage. After adjustment of the pulser frequency, ions as high in m/z as 400,000 were detected. Significant losses in efficiency were noted above m/z 250,000 that is likely due to roll-over in the ion detector efficiency and possibly also due to limitations in ion transfer efficiency from the collision quadrupole to the pulser region of the mass analyzer. No measurable decrease in the apparent mass resolving power was noted upon charge state reduction of the model ions. Resonance ejection techniques that employ the dipolar AC capabilities of the quadrupoles allow for ion isolation at m/z values much greater than the RF/DC limitation of Q1 of m/z = 2100. For example, at the highest low-mass cutoff achievable in the collision quadrupole (m/z = 500), it is possible to isolate ions of m/z as high as 62,000. This is limited by the lowest dipolar AC frequency (5 kHz) that can be applied. A simple model is included to provide for an estimate of the ion cloud radius based on ion m/z, ion z, and ion trap operating conditions. The model predicts that singly charged ions of 1 MDa and thermal energy can be contained in the ion trap at the maximum low-mass cutoff, although such an ion would not be detected efficiently. Doubly charged GroEL ions were observed experimentally. Collectively, the performance characteristics at high m/z, the functionality provided by the standard instrument capabilities, the modifications described above, and highly flexible instrument control software provide for a highly versatile platform for the study of high mass ion/ion reactions.

Valet Parking for Protein Ion Charge State Concentration: Ion/Molecule Reactions in Linear Ion Traps.

There are several analytical applications in which it is desirable to concentrate analyte ions generated over a range of charge states into a single charge state. This has been demonstrated in the gas phase via ion/ion reactions in conjunction with a technique termed ion parking, which can be implemented in electrodynamic ion traps. Ion parking depends upon the selective inhibition of the reaction of a selected charge state or charge states. In this work, we demonstrate a similar charge state concentration effect using ion/molecule reactions rather than ion/ion reactions. The rates of ion/molecule reactions cannot be affected in the manner used in conventional ion parking. Rather, to inhibit the progression of ion/molecule proton transfer reactions, the product ions must be removed from the reaction cell as they are formed and transferred to an ion trap where no reactions occur. This is accomplished here with mass-selective axial ejection (MSAE) from one linear ion trap to another. The application of MSAE to inhibit ion/molecule reactions is referred to as "valet parking" as it entails the transport of the ions of interest to a remote location for storage. Valet parking is demonstrated using model proteins to concentrate ion signal dispersed over multiple charge states into largely one charge state. Additionally, it has been applied to a simple two-protein mixture of cytochrome c and myoglobin.

A Miniaturized Fourier Transform Electrostatic Linear Ion Trap Mass Spectrometer: Mass Range and Resolution.

Mass resolution (M/ΔMFWHM) was increased by reducing the axial length of a Fourier transform electrostatic linear ion trap (FT-ELIT) mass spectrometer. The increase in mass resolution corresponds directly to increased axial ion frequencies in the FT-ELIT. Increased mass resolution was demonstrated for equivalent transient lengths in a 5.25″ versus 2.625″ ELIT using the isotopes of [bradykinin+2H]2+ and [insulin+5H]5+ as test ions. Both bradykinin and insulin show mass resolution increases of ~ 90% allowing baseline resolution of the [insulin+5H]5+ isotopes after only 300 ms of data acquisition. Relative changes in mass/charge range were explored using mirror switching to trap ions injected axially into the ELIT. When trapping ions using mirror switching, the mass/charge range in a FT-ELIT mass spectrometer for a given switch time is determined by the time required for fast ions to enter and exit the trap after one reflection versus the time it takes for slow ions to enter the trap. By reducing the length of the FT-ELIT mass spectrometer while maintaining a constant distance from the point from which ions are initially accelerated to the entrance mirror, only the low m/z limit is affected for a given mirror switching time. For the two ELIT lengths examined here, the effective mass/charge range at any given switch time is reduced from m/zlow-8.9*m/zlow for the 5.25″ ELIT to m/zlow-5.2*m/zlow for the 2.625″ ELIT. Graphical Abstract.