Full Separation and Sequencing of Isomeric Proteoforms in the Middle-Down Mass Range Using Cyclic Ion Mobility and Electron Capture Dissociation.

Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.

Toward Rapid Aspartic Acid Isomer Localization in Therapeutic Peptides Using Cyclic Ion Mobility Mass Spectrometry.

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

High-Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp-substrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein.

HBc, the capsid-forming "core protein" of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry-mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.

The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages.

The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21(1-552) (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21(1-552) trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21(1-552) trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21(1-552) are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.