Implementing Digital-Waveform Technology for Extended m/ z Range Operation on a Native Dual-Quadrupole FT-IM-Orbitrap Mass Spectrometer

Abstract

Here, we describe a digital-waveform dual-quadrupole mass spectrometer that enhances the performance of our drift tube FT-IMS high-resolution Orbitrap mass spectrometer (MS). The dual-quadrupole analyzer enhances the instrument capabilities for studies of large protein and protein complexes. The first quadrupole (q) provides a means for performing low-energy collisional activation of ions to reduce or eliminate noncovalent adducts, viz., salts, buffers, detergents, and/or endogenous ligands. The second quadrupole (Q) is used to mass-select ions of interest for further interrogation by ion mobility spectrometry and/or collision-induced dissociation (CID). Q is operated using digital-waveform technology (DWT) to improve the mass selection compared to that achieved using traditional sinusoidal waveforms at floated DC potentials (>500 V DC). DWT allows for increased precision of the waveform for a fraction of the cost of conventional RF drivers and with readily programmable operation and precision (Hoffman, N. M. . A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

Keywords: Fourier-transform ion mobility-Orbitrap mass spectrometry; digital-waveform technology; native mass spectrometry; quadrupole mass spectrometry.

Figure 1.

SolidWorks rendering of the nano-ESI dual-quadrupole FT-IM-PF-DT coupled to the HCD cell of a Thermo Exactive Plus Orbitrap with Extended Mass Range (qQ-FT-IM-PF-DT-HCD Orbitrap) with the major components labeled and the applied DC potential gradient across the instrument. The length of the DT is not to scale in the rendering, i.e., the vacuum box and qQ are enlarged to illustrate this device better; the DT, 8-pole, and other ion optics have been described previously.

Figure 2.

(A) Total ion current (TIC) detected as the frequency of Q (85/15 duty cycle with a ±10 V DC bias applied on rod pairs at 300 V_pp) is increased from 75 to 125 kHz. Frequency stepped in 1 kHz steps at the rate of 1 kHz per second in panel A to produce an m/z selection chromatogram of quadrupole 2 (Q). (B) Extracted MS for the given frequency range shown in panel A. Full-ion transmission and isolation of a single charge state for CRP (panel C), AmtB (panel D), and GroEL (panel E).

Figure 3.

Mass spectra illustrating how mild collisional activation in the q of the qQ-FT-IM-PF-DT Orbitrap can be used to remove adducted species (salts, endogenous lipids, and other small molecules) from (A) the trimeric ammonia transport channel (AmtB, 127 kDa) and (B) GroEL (801 kDa, tetradecamer (n = 14)). The collision voltages indicated refer to the potential differences between the exit of the ion funnel and the time-averaged DC potential of the activation quad, q at various DC potential drops. Collisional activation in q may produce non-native state ions; whether this occurs or not can be assessed by using ion mobility, as shown in panels B and C of Figure 4. The resolution (denoted R based on fwhm) for the most abundant charge state is listed in each panel. The insets in (B) more clearly illustrate the higher resolving power following collisional activation in q for the GroEL tetradecamer.

Figure 4.

Mass spectra and IMS arrival-time distribution plots for (A) pentamer and decamer complexes of C-reactive protein (CRP) and (B) the tetradecamer GroEL chaperone.

Figure 5.

Complex-down characterization of the CRP. (A) The native MS spectrum of CPR is shown in the top panel, and each of the panels below contains mass spectra resulting from collisional activation (at the indicated voltage) of the ion populations in the panel above. Panel (B) contains the mass spectrum obtained by using DWT to mass-select the CRP monomer (24⁺ pentamer ion), and panel (C) contains the top-down mass spectrum of the mass-selected ion shown in panel (B). The amino acid sequence of CRP is shown, and the sequence informative fragment ion is labeled accordingly in panel (D).