Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6–1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (−3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

Mass spectrometry is a central technology in the life sciences, providing our most comprehensive account of the molecular inventory of the cell. In parallel with developments in mass spectrometry technologies targeting such assessments of cellular composition, mass spectrometry tools have emerged as versatile probes of biomolecular stability. In this review, we cover recent advancements in this branch of mass spectrometry that target proteins, a centrally important class of macromolecules that accounts for most biochemical functions and drug targets. Our efforts cover tools such as hydrogen–deuterium exchange, chemical cross-linking, ion mobility, collision induced unfolding, and other techniques capable of stability assessments on a proteomic scale. In addition, we focus on a range of application areas where mass spectrometry-driven protein stability measurements have made notable impacts, including studies of membrane proteins, heat shock proteins, amyloidogenic proteins, and biotherapeutics. We conclude by briefly discussing the future of this vibrant and fast-moving area of research.

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.

Cerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.

The versatile pyridyl synthons of mixed N,Se donor ligands bis(pyridin-2-ylmethyl)selane (L) and 1,2bis(pyridin-2-ylmethyl)diselane (L*) served the dual roles of metal chelating anchors and mediators of supramolecular associations in a crystallographic approach to documenting uncommon bonding between divalent Group 12 metal ions and neutral selenium functionalities. Five new molecular species with diverse ligand configurations and conformations were structurally characterized. Two macrocyclic polymorphs of [ZnLCl 2 ] have intermolecular Se−Cl interactions rather than the desired intramolecular Zn−Se binding. Rare examples of Cd(II)-selenoether bonding are provided by tricoordinate L in dinuclear [CdLCl(μ-Cl)] 2 and mononuclear [CdL(μ-NO 3 ) 2 ]. Unique intramolecular Zn-selenoether and Cd-diselenide bonds are reported for [ZnL(μ-NO 3 ) 2 ] and [CdL*(μ-NO 3 ) 2 ], respectively. In addition, a cocrystal of [CdL(μ-NO 3 ) 2 ] and [CdL*(μ-NO 3 ) 2 ] is structurally characterized. The intermolecular pyridyl π-interactions observed in these diverse structures are highlighted.