Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer.

Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established. Here we evaluate the reproducibility of the CIU data produced across three laboratories (University of Michigan, Texas A&M University, and Vanderbilt University). CIU data were collected for a variety of protein ions ranging from 8.6-66 kDa. Within the same laboratory, the CIU fingerprints were found to be repeatable with root mean square deviation (RMSD) values of less than 5%. Collision cross section (CCS) values of the CIU intermediates were consistent across the laboratories, with most features exhibiting an interlaboratory reproducibility of better than 1%. In contrast, the activation potentials required to induce protein CIU transitions varied between the three laboratories. To address these differences, three source assemblies were constructed with an updated ion activation hardware design utilizing higher mechanical tolerance specifications. The production-grade assemblies were found to produce highly consistent CIU data for intact antibodies, exhibiting high precision ion CCS and CIU transition values, thus opening the door to establishing databases of CIU fingerprints to support future biomolecular classification efforts.

THE IMS PARADOX: A PERSPECTIVE ON STRUCTURAL ION MOBILITY-MASS SPECTROMETRY.

Studies of large proteins, protein complexes, and membrane protein complexes pose new challenges, most notably the need for increased ion mobility (IM) and mass spectrometry (MS) resolution. This review covers evolutionary developments in IM-MS in the authors' and key collaborators' laboratories with specific focus on developments that enhance the utility of IM-MS for structural analysis. IM-MS measurements are performed on gas phase ions, thus "structural IM-MS" appears paradoxical-do gas phase ions retain their solution phase structure? There is growing evidence to support the notion that solution phase structure(s) can be retained by the gas phase ions. It should not go unnoticed that we use "structures" in this statement because an important feature of IM-MS is the ability to deal with conformationally heterogeneous systems, thus providing a direct measure of conformational entropy. The extension of this work to large proteins and protein complexes has motivated our development of Fourier-transform IM-MS instruments, a strategy first described by Hill and coworkers in 1985 (Anal Chem, 1985, 57, pp. 402-406) that has proved to be a game-changer in our quest to merge drift tube (DT) and ion mobility and the high mass resolution orbitrap MS instruments. DT-IMS is the only method that allows first-principles determinations of rotationally averaged collision cross sections (CSS), which is essential for studies of biomolecules where the conformational diversities of the molecule precludes the use of CCS calibration approaches. The Fourier transform-IM-orbitrap instrument described here also incorporates the full suite of native MS/IM-MS capabilities that are currently employed in the most advanced native MS/IM-MS instruments. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Development of Native MS Capabilities on an Extended Mass Range Q-TOF MS.

Native mass spectrometry (nMS) is increasingly used for studies of large biomolecules (>100 kDa), especially proteins and protein complexes. The growth in this area can be attributed to advances in native electrospray ionization as well as instrumentation that is capable of accessing high mass-to-charge (m/z) regimes without significant losses in sensitivity and resolution. Here, we describe modifications to the ESI source of an Agilent 6545XT Q-TOF MS that is tailored for analysis of large biomolecules. The modified ESI source was evaluated using both soluble and membrane protein complexes ranging from ~127 to ~232 kDa and the ~801 kDa protein chaperone GroEL. The increased mass resolution of the instrument affords the ability to resolve small molecule adducts and analyze collision-induced dissociation products of the native complexes.

First-Principles Collision Cross Section Measurements of Large Proteins and Protein Complexes.

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.

Topological Characterization of Coordination-Driven Self-assembly Complexes: Applications of Ion Mobility-Mass Spectrometry.

Coordination-driven self-assembly (CDSA) is increasingly used to synthesize coordination complexes containing metal-centered electron acceptors and typically nitrogen-containing electron donors. Characterization of the structures obtained from CDSA via crystallographic or spectroscopic means is limited due to difficulties in forming single crystals for X-ray studies and overlapping precursor and product signals in NMR. Here, we employ ion mobility-mass spectrometry (IM-MS), which provides a direct measure of size and shape of the CDSA complexes, to study the intact reaction products of a rhomboid-shaped complex. This approach negates the need for product isolation and crystallization and allows for tracking of the product distribution as a function of time. A potential challenge of IM-MS is that the size/shape of the observed CDSA complexes can vary with internal energy; however, we show that proper tuning of the instrument reduces the effects of collisional activation thereby allowing for retention of ion conformations that reflect solution-phase ion structures. Graphical Abstract.