Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P.

tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.

Collision-Induced Unfolding Reveals Disease-Associated Stability Shifts in Mitochondrial Transfer Ribonucleic Acids.

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing their applications in biotechnology. The inherent dynamism of RNAs creates numerous obstacles in capturing their biologically relevant higher-order structures (HOSs), and as a result, many RNA functions remain unknown. In this study, we describe the development of native ion mobility-mass spectrometry and collision-induced unfolding (CIU) for the structural characterization of a variety of RNAs. We evaluate the ability of these techniques to preserve native structural features in the gas phase across a wide range of functional RNAs. Finally, we apply these tools to study the elusive mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNAs via the correlated complexity of CIU fingerprints and RNA HOS, the observation of predicted stability shifts in the control RNA samples, and the retention of predicted magnesium binding events in gas-phase RNA ions. Significant differences in collision cross section and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial tRNA maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization.

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers.

Sensitive analytical techniques that are capable of detecting and quantifying disease-associated biomolecules are indispensable in our efforts to understand disease mechanisms and guide therapeutic intervention through early detection, accurate diagnosis, and effective monitoring of disease. Parkinson's Disease (PD), for example, is one of the most prominent neurodegenerative disorders in the world, but the diagnosis of PD has primarily been based on the observation of clinical symptoms. The protein α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of α-syn has prevented its widespread use as a biomarker. Antibody-based methods such as immunoassays and mass spectrometry-based approaches have been used to measure a limited number of α-syn forms; however, these methods fail to differentiate variants of α-syn that display subtle differences in only the sequence and structure. In this work, we developed a cyclic ion mobility-mass spectrometry method that combines multiple stages of activation and timed ion selection to quantify α-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several α-syn variants present at physiological levels in biological fluid. Taken together, this approach can be used to galvanize future efforts aimed at understanding the underlying mechanisms of PD and serves as a starting point for the development of future protein-structure-based diagnostics and therapeutic interventions.

Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding Rapidly Characterize the Structural Polydispersity and Stability of an Fc-Fusion Protein.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis.

INTRODUCTION: The pharmaceutical industry continues to expand its search for innovative biotherapeutics. The comprehensive characterization of such therapeutics requires many analytical techniques to fully evaluate critical quality attributes, making analysis a bottleneck in discovery and development timelines. While thorough characterization is crucial for ensuring the safety and efficacy of biotherapeutics, there is a need to further streamline analytical characterization and expedite the overall timeline from discovery to market. AREAS COVERED: This review focuses on recent developments in liquid-phase separations coupled with ion mobility-mass spectrometry (IM-MS) for the development and characterization of biotherapeutics. We cover uses of IM-MS to improve the characterization of monoclonal antibodies, antibody-drug conjugates, host cell proteins, glycans, and nucleic acids. This discussion is based on an extensive literature search using Web of Science, Google Scholar, and SciFinder. EXPERT OPINION: IM-MS has the potential to enhance the depth and efficiency of biotherapeutic characterization by providing additional insights into conformational changes, post-translational modifications, and impurity profiles. The rapid timescale of IM-MS positions it well to enhance the information content of existing assays through its facile integration with standard liquid-phase separation techniques that are commonly used for biopharmaceutical analysis.

Mass Spectrometry Methods for Measuring Protein Stability.

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.

BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Ion mobility-mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy.

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine-Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility-mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft-mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids.

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson's disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Characterization of a [4Fe-4S]-dependent LarE sulfur insertase that facilitates nickel-pincer nucleotide cofactor biosynthesis in Thermotoga maritima.

Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarETm) and show that it uses these three conserved cysteine residues to bind a [4Fe-4S] cluster that is required for sulfur transfer. Notably, we found LarETm retains all side chain sulfur atoms, in contrast to LarELp. We also demonstrate that when provided with L-cysteine and cysteine desulfurase from Escherichia coli (IscSEc), LarETm functions catalytically with IscSEc transferring sulfane sulfur atoms to LarETm. Native mass spectrometry results are consistent with a model wherein the enzyme coordinates sulfide at the nonligated iron atom of the [4Fe-4S] cluster, forming a [4Fe-5S] species, and transferring the noncore sulfide to the activated substrate. This proposed mechanism is like that of TtuA that catalyzes sulfur transfer during 2-thiouridine synthesis. In conclusion, we found that LarE sulfur insertases associated with NPN biosynthesis function either by sacrificial sulfur transfer from the protein or by transfer of a noncore sulfide bound to a [4Fe-4S] cluster.

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain.

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.

Infrared Photoactivation Enables Improved Native Top-Down Mass Spectrometry of Transmembrane Proteins.

Membrane proteins are often challenging targets for native top-down mass spectrometry experimentation. The requisite use of membrane mimetics to solubilize such proteins necessitates the application of supplementary activation methods to liberate protein ions prior to sequencing, which typically limits the sequence coverage achieved. Recently, infrared photoactivation has emerged as an alternative to collisional activation for the liberation of membrane proteins from surfactant micelles. However, much remains unknown regarding the mechanism by which IR activation liberates membrane protein ions from such micelles, the extent to which such methods can improve membrane protein sequence coverage, and the degree to which such approaches can be extended to support native proteomics. Here, we describe experiments designed to evaluate and probe infrared photoactivation for membrane protein sequencing, proteoform identification, and native proteomics applications. Our data reveal that infrared photoactivation can dissociate micelles composed of a variety of detergent classes, without the need for a strong IR chromophore by leveraging the relatively weak association energies of such detergent clusters in the gas phase. Additionally, our data illustrate how IR photoactivation can be extended to include membrane mimetics beyond micelles and liberate proteins from nanodiscs, liposomes, and bicelles. Finally, our data quantify the improvements in membrane protein sequence coverage produced through the use of IR photoactivation, which typically leads to membrane protein sequence coverage values ranging from 40 to 60%.

Development of an Automated, High-Throughput Methodology for Native Mass Spectrometry and Collision-Induced Unfolding.

Native ion mobility mass spectrometry (nIM-MS) has emerged as a useful technology for the rapid evaluation of biomolecular structures. When combined with collisional activation in a collision-induced unfolding (CIU) experiment, nIM-MS experimentation can be leveraged to gain greater insight into biomolecular conformation and stability. However, nIM-MS and CIU remain throughput limited due to nonautomated sample preparation and introduction. Here, we explore the use of a RapidFire robotic sample handling system to develop an automated, high-throughput methodology for nMS and CIU. We describe native RapidFire-MS (nRapidFire-MS) capable of performing online desalting and sample introduction in as little as 10 s per sample. When combined with CIU, our nRapidFire-MS approach can be used to collect CIU fingerprints in 30 s following desalting by using size exclusion chromatography cartridges. When compared to nMS and CIU data collected using standard approaches, ion signals recorded by nRapidFire-MS exhibit identical ion collision cross sections, indicating that the same conformational populations are tracked by the two approaches. Our data further suggest that nRapidFire-MS can be extended to study a variety of biomolecular classes, including proteins and protein complexes ranging from 5 to 300 kDa and oligonucleotides. Furthermore, nRapidFire-MS data acquired for biotherapeutics suggest that nRapidFire-MS has the potential to enable high-throughput nMS analyses of biopharmaceutical samples. We conclude by discussing the potential of nRapidFire-MS for enabling the development of future CIU assays capable of catalyzing breakthroughs in protein engineering, inhibitor discovery, and formulation development for biotherapeutics.

Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding of Designed Bispecific Antibody Therapeutics.

Bispecific antibodies (bsAbs) represent a critically important class of emerging therapeutics capable of targeting two different antigens simultaneously. As such, bsAbs have been developed as effective treatment agents for diseases that remain challenging for conventional monoclonal antibody (mAb) therapeutics to access. Despite these advantages, bsAbs are intricate molecules, requiring both the appropriate engineering and pairing of heavy and light chains derived from separate parent mAbs. Current analytical tools for tracking the bsAb construction process have demonstrated a limited ability to robustly probe the higher-order structure (HOS) of bsAbs. Native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) have proven to be useful tools in probing the HOS of mAb therapeutics. In this report, we describe a series of detailed and quantitative IM-MS and CIU data sets that reveal HOS details associated with a knob-into-hole (KiH) bsAb model system and its corresponding parent mAbs. We find that quantitative analysis of CIU data indicates that global KiH bsAb stability occupies an intermediate space between the stabilities recorded for its parent mAbs. Furthermore, our CIU data identify the hole-containing half of the KiH bsAb construct to be the least stable, thus driving much of the overall stability of the KiH bsAb. An analysis of both intact bsAb and enzymatic fragments allows us to associate the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. This result is likely general for CIU data collected for low charge state mAb ions and is supported by data acquired for deglycosylated KiH bsAb and mAb constructs, each of which indicates greater destabilization of the second CIU transition observed in our data. When integrated, our CIU analysis allows us to link changes in the first CIU transition primarily to the Fab region of the hole-containing halfmer, while the second CIU transition is likely strongly connected to the Fc region of the knob-containing halfmer. Taken together, our results provide an unprecedented road map for evaluating the domain-level stabilities and HOS of both KiH bsAb and mAb constructs using CIU.

High-Throughput Liquid Chromatographic Analysis Using a Segmented Flow Injector with a 1 s Cycle Time.

High-throughput screening (HTS) workflows are revolutionizing many fields, including drug discovery, reaction discovery and optimization, diagnostics, sensing, and enzyme engineering. Liquid chromatography (LC) is commonly deployed during HTS to reduce matrix effects, distinguish isomers, and preconcentrate prior to detection, but LC separation time often limits throughput. Although subsecond LC separations have been demonstrated, they are rarely utilized during HTS due to limitations associated with the speed of common autosamplers. In this work, these limits are overcome by utilizing droplet microfluidics for sample introduction. In the method, a train of samples segmented by air are continuously pumped into the inlet of an LC injection valve that is actuated once each sample fills the sample loop. Coupled with 2.1 mm diameter × 5 mm long columns packed with 2.7 µm superficially porous C18 particles operated at 5 mL/min, the injector enabled separation of 3 components at 1 s/sample and analysis of a 96-well plate in 1.6 min with <2% peak area relative standard deviation. Analyte-dependent carryover was minimized by including wash droplets composed of organic solvent in between sample droplets. High-throughput LC coupled with mass spectrometric detection using the segmented flow injector was applied to a screen of inhibitors of a cytochrome P450-catalyzed hydroxylation reaction. Measurements of the reaction substrate and product concentrations made using fast LC with the segmented flow injector correlated well with measurements made using a more conventional, 3 min LC method. These results demonstrate the potential for droplet microfluidics to be used for sample introduction during high-throughput LC analysis.

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.

Collision Cross Sections for Native Proteomics: Challenges and Opportunities.

Recent advancements place a comprehensive catalog of protein structure, oligomeric state, sequence, and modification status tentatively within reach, thus providing an unprecedented roadmap to therapies for many human diseases. To achieve this goal, revolutionary technologies capable of bridging key gaps in our ability to simultaneously measure protein composition and structure must be developed. Much of the current progress in this area has been catalyzed by mass spectrometry (MS) tools, which have become an indispensable resource for interrogating the structural proteome. For example, methods associated with native proteomics seek to comprehensively capture and quantify the endogenous assembly states for all proteins within an organism. Such technologies have often been partnered with ion mobility (IM) separation, from which collision cross section (CCS) information can be rapidly extracted to provide protein size information. IM technologies are also being developed that utilize CCS values to enhance the confidence of protein identification workflows derived from liquid chromatography-IM-MS analyses of enzymatically produced peptide mixtures. Such parallel advancements in technology beg the question: can CCS values prove similarly useful for the identification of intact proteins and their complexes in native proteomics? In this perspective, I examine current evidence and technology trends to explore the promise and limitations of such CCS information for the comprehensive analysis of multiprotein complexes from cellular mixtures.

Ion Mobility-Mass Spectrometry Coupled to Droplet Microfluidics for Rapid Protein Structure Analysis and Drug Discovery.

Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by ∼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis.

Ion Mobility-Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates.

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1ß. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM-MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1ß, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1ß monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

SERF engages in a fuzzy complex that accelerates primary nucleation of amyloid proteins.

The assembly of small disordered proteins into highly ordered amyloid fibrils in Alzheimer's and Parkinson's patients is closely associated with dementia and neurodegeneration. Understanding the process of amyloid formation is thus crucial in the development of effective treatments for these devastating neurodegenerative diseases. Recently, a tiny, highly conserved and disordered protein called SERF was discovered to modify amyloid formation in Caenorhabditis elegans and humans. Here, we use kinetics measurements and native ion mobility-mass spectrometry to show that SERF mainly affects the rate of primary nucleation in amyloid formation for the disease-related proteins Aß40 and α-synuclein. SERF's high degree of plasticity enables it to bind various conformations of monomeric Aß40 and α-synuclein to form structurally diverse, fuzzy complexes. This structural diversity persists into early stages of amyloid formation. Our results suggest that amyloid nucleation is considerably more complex than age-related conversion of Aß40 and α-synuclein into single amyloid-prone conformations.