capillary-electrophoresis-based-hydrogendeuterium-exchange-for

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

nanjing normal university

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry.

Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.

Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry.

Direct mapping of protein disulfide patterns using top-down mass spectrometry (MS) is often hampered by inadequate fragmentation at the disulfide-enclosing region, and insufficient structural information provided by the fragments. Here we used electron-transfer/high energy collision dissociation (EThcD) to improve the fragmentation efficiency, and developed strategies that minimize the false positive identification of fragments and deconvolute the signals representing specific modifications made to the disulfide-cleavage-induced fragments. We observed clear correlations between unique modification (attachment or removal of H or SH) patterns and the number of disulfide bonds that enclose the corresponding region. Using the characteristic signature fragments, we in part localized the Cys-bridging sites in disulfide-scrambled lysozymes, and reduced the number of putative disulfide patterns from 104 to 6. The results demonstrated the feasibility of direct analysis of complex disulfide patterns using top-down MS.

Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry.

Native mass spectrometry (MS) is an emerging approach for characterizing biomacromolecular structure and interactions under physiologically relevant conditions. In native MS measurement, intact macromolecules or macromolecular complexes are directly ionized from a non-denaturing solvent, and key noncovalent interactions that hold the complexes together can be preserved for MS analysis in the gas phase. This technique provides unique multi-level structural information such as conformational changes, stoichiometry, topology and dynamics, complementing conventional biophysical techniques. Despite the maturation of native MS and greatly expanded range of applications in recent decades, further dissemination is needed to make the community aware of such a technique. In this review, we attempt to provide an overview of the current body of knowledge regarding major aspects of native MS and explain how such technique contributes to the characterization of biomacromolecular higher-order structure and interactions.

How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions?

Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF-MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF-MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF-MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights ( M) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact M values showed ppm level accuracy compared to theoretical predictions.

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface.

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States.

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Higher order structure of protein therapeutics is an important quality attribute, which dictates both potency and safety. While modern experimental biophysics offers an impressive arsenal of state-of-the-art tools that can be used for the characterization of higher order structure, many of them are poorly suited for the characterization of biopharmaceutical products. As a result, these analyses were traditionally carried out using classical techniques that provide relatively low information content. Over the past decade, mass spectrometry made a dramatic debut in this field, enabling the characterization of higher order structure of biopharmaceuticals as complex as monoclonal antibodies at a level of detail that was previously unattainable. At present, mass spectrometry is an integral part of the analytical toolbox across the industry, which is critical not only for quality control efforts, but also for discovery and development.

Mass spectrometry-based methods in characterization of the higher order structure of protein therapeutics.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase.

Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of , effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG):C1q complexes and prevents downstream complement activation on the surface of  Finally, we demonstrate that IgG3 antibodies against  can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.

Article	Year
Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry. Analytical chemistry\| PubMed ID: 28383250	2017
Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry. The Analyst\| PubMed ID: 29303166	2018
Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry. The Analyst\| PubMed ID: 29362732	2018
How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions? Methods (San Diego, Calif.)\| PubMed ID: 29704661	2018
High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface. Analytical chemistry\| PubMed ID: 29993237	2018
Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States. Analytical chemistry\| PubMed ID: 30830762	2019
Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces. Nano letters\| PubMed ID: 31184907	2019
RNA sequencing by direct tagmentation of RNA/DNA hybrids. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31988135	2020
Mass spectrometry-based methods in characterization of the higher order structure of protein therapeutics. Journal of pharmaceutical and biomedical analysis\| PubMed ID: 32092629	2020
Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase. Analytical chemistry\| PubMed ID: 33211472	2020
Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33563762	2021

Guanbo Wang

Biography

JoVE Journal Publications

Other Publications