Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.

Experimental Evaluation of Higher Order Stability Zones Using a Digitally Operated Quadrupole Mass Filter.

Recent improvements to the comparison-based method of digital waveform generation increased the reproducibility of the waveforms so that the higher-order Mathieu stability zones can be accessed reliably. Digitally driven quadrupole mass filters access these zones using a fixed AC voltage and rectangular waveforms that are defined by a duty cycle. In this context, the duty cycle is the fraction of the waveform period where the waveform remains in the high state. Because digitally driven quadrupoles navigate stability using a duty cycle, there is no need to apply a resolving DC offset between electrode pairs. Accessing the higher stability zones using a conventional resonantly tuned RF requires the use of thousands of AC and DC voltages making the mode of operation less accessible with these devices. Stability zones higher than (1,1) and (2,1) have theoretical resolving powers that are on the order 1,140 and 3,447 at fwhm which drives efforts to practically access these operational conditions. Accessing these zones digitally requires the use of extremely precise waveforms. In a previous effort, waveform generation produced waveforms to reliably access the (1,1) and (2,1) zones without impacting performance. However, recent work found more improvement was needed to reliably access neighboring higher stability zones. Derived from that work, it was determined that a waveform resolution of ∼10 ppm or less was needed to reliably access the (3,1) and (3,2) zones. The present work utilized digital waveforms that achieve this level of precision to experimentally access and characterize attributes of the (3,1) and (3,2) zones. This work dives into the investigation of different beam energies to overcome the destabilizing fringing fields, improve transmission, and their overall effect on the experimental resolving power and signal-to-noise. In addition, the AC voltage of the driving RF was varied to understand the effects on the initial ion beam energy that is needed to achieve balanced separation and how the overall signal-to-noise is affected. Lastly, an assessment was made on the effects of the temporal parameters of a digital mass scan on peak sensitivity, peak fidelity, and overall duration for a scan.

Effect of Jitter on Digital Mass Filter Analysis in Higher Stability Zones.

Waveform reproducibility is a critical factor for performing high resolution mass analysis with digitally operated quadrupole mass filters and traps operating in higher stability zones. In this work, Hill equation-based stability calculations were used to define the effect of period jitter on mass analysis in higher stability zones. These calculations correlate well with experimental observations in higher stability zones. Comparison of experiment to theory supplies the basis for defining jitter-based expectations and limits for mass analysis in higher zones.

Experimental Validation of the Digital Tandem Mass Filter.

This work presents the experimental evaluation of a digital tandem mass filter that is composed of two digitally operated low-resolution mass filters in series whose mass windows are shifted with respect to each other. The overlap of the mass windows allows the resolution (Δm) of ions to be narrowed to provide better resolving power, while the acceptance of the tandem mass filter is defined by the acceptance of the first low-resolution quadrupole. Our experiments show that digital operation fulfills the promise of the tandem mass filter for providing better ion transmission at the same or better resolving power as a single quadrupole mass filter. It allows the user to continuously adjust the resolving power and sensitivity to meet current needs. Most importantly, the observed resolving power/sensitivity characteristics are the same at any mass and m/z.

Will the Digital Mass Filter Be the Next High-Resolution High-Mass Analyzer?

Mass filter operation in higher stability zones is known to provide better resolution. Unfortunately, for sine driven instruments, higher stability zone operation reduces the accessible mass range and increases the degenerative effects of fringe fields. Conversely, digitally driven mass filters do not suffer from loss of mass range, and the fringe field effects do not increase significantly by switching stability zones because the AC voltage is always constant and the DC voltage is always zero. This work catalogues 12 stability zones that are accessible with the new digital waveform generation technology. These zones have theoretical baseline resolving powers that range from 22 to 1 300 000 with pseudopotential well depths that range from 3.5 to 43 V. Operation in higher stability zones also has the advantage of aligned axial stability wells. That alignment maximizes the pseudopotential well depth for each higher stability zone, making them more than an order of magnitude greater than the standard ∼0.2 V well of a sine filter operating in the first stability zone at unit resolution. Increased pseudopotential well depth correlates with better ion transmission and sensitivity. Our theoretical examination suggests that the digital mass filter can obtain both high resolution and high sensitivity with essentially unlimited mass range.