Absorption Mode Fourier Transform Ion Mobility Mass Spectrometry Multiplexing Combined with Half-Window Apodization Windows Improves Resolution and Shortens Acquisition Times

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Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.

Section: Methodsmentioning

confidence: 99%

Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry

Sipe

Fouque

Garabedian

et al. 2022

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“…All the experiments described below employ the DWT operation of the Q MS. Ions exiting Q enter an octupole ion guide (47 mm) and then transfer to a 55 cm PF-DT maintained at pressures of 1–2 Torr of He. The DT-IMS is operated in the FT-IMS mode as described previously. ,− The ions exiting the DT enter a final octupole ion guide and are transferred to the HCD cell of the Orbitrap mass spectrometer, as described previously …”

Section: Methodsmentioning

confidence: 99%

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

McCabe

Jones

Walker

et al. 2021

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. et al.. A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

“…While Fourier-based multiplexing of ion mobility experiments has seen success, ,− there are ambiguities that exist within the experiment, particularly the assignment of frequencies at a given time point within the frequency-modulated signal. In short, the traditional method of performing FT-IMS experiments requires synchronizing only the start and end times of the frequency sweep and first and last scans of the mass spectrometer.…”

Section: Introductionmentioning

confidence: 99%

Synchronized Stepped Frequency Modulation for Multiplexed Ion Mobility Measurements

Cabrera

Clowers

2022

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Implementation of frequency-encoded multiplexing for ion mobility spectrometry (e.g., Fourier transform ion mobility spectrometry (FT-IMS)) has facilitated the direct coupling of drift tube ion mobility instrumentation with ion-trap mass analyzers despite their duty cycle mismatch. Traditionally, FT-IMS experiments have been carried out to utilize continuous linear frequency sweeps that are independent of the scan rate of the ion-trap mass analyzer, thus creating a situation where multiple frequencies are swept over two sequential mass scans. This in turn creates a degree of ambiguity in which the ion current derived from a single modulation frequency cannot be assigned to a single data point in the frequency-modulated signal. In an effort to eliminate this ambiguity, this work describes a discrete stepwise function to modulate the ion gates of the IMS while synchronization between the generated frequencies and the scan rate of the linear ion trap is achieved. While the number of individual frequencies used in the stepped frequency sweeps is less than in continuous linear modulation experiments, there is no loss in performance and high levels of precision are maintained across differing combinations of terminal frequencies and scan lengths. Furthermore, the frequency-scan synchronization enables further data-processing techniques such as linear averaging of the frequency modulated signal to drastically improve signal-to-noise ratio for both high and low intensity analytes.