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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).

Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ~5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ~60 is obtained for pyruvate kinase (MW ~ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ~ 68 kDa) is less than ~20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.

The amyloidogenic mechanism of transthyretin is still debated but understanding it fully could lend insight into disease progression and potential therapeutics. Transthyretin was investigated revealing a metal-induced (Cr/Cu) oxidation pathway leading to N-terminal backbone fragmentation and oligomer formation; previously hidden details were revealed only by FT-IM-Orbitrap MS and surface-induced dissociation.