Native Top-down Mass Spectrometry Research Articles

A Conformation-Specific Approach to Native Top-down Mass Spectrometry.

Native top-down mass spectrometry is a powerful approach for characterizing proteoforms and has recently been applied to provide similarly powerful insights into protein conformation. Current approaches, however, are limited such that structural insights can only be obtained for the entire conformational landscape in bulk or without any direct conformational measurement. We report a new ion-mobility-enabled method for performing native top-down MS in a conformation-specific manner. Our approach identified conformation-linked differences in backbone dissociation for the model protein calmodulin, which simultaneously informs upon proteoform variations and provides structural insights. We also illustrate that our method can be applied to protein-ligand complexes, either to identify components or to probe ligand-induced structural changes.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methods─namely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociation─in their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Investigating the Intramolecular Competition of Different RNA Binding Motifs for Neomycin B by Native Top-Down Mass Spectrometry.

The ongoing search for small molecule drugs that target ribonucleic acids (RNA) is complicated by a limited understanding of the principles that govern RNA-small molecule interactions. Here we have used stoichiometry-resolved native top-down mass spectrometry (MS) to study the binding of neomycin B to small model hairpin RNAs, an unstructured RNA, and a viral RNA construct. For 15-22 nt model RNAs with hairpin structure, we found that neomycin B binding to hairpin loops relies on interactions with both the nucleobases and the 2'-OH groups, and that a simple 5' or 3' overhang can introduce an additional binding motif. For a 47 nt RNA construct derived from stem IA of the human immunodeficiency virus 1 (HIV-1) rev response element (RRE) RNA, native top-down MS identified four different binding motifs, of which the purine-rich internal loop showed the highest affinity for neomycin B. Stoichiometry-resolved binding site mapping by native top-down MS allows for a new perspective on binding specificity, and has the potential to reveal unexpected principles of small molecule binding to RNA.

Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca2+) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.

Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present challenges to study using conventional structural biology techniques. Here we develop a native nanoproteomics strategy for the enrichment and subsequent native top-down mass spectrometry (nTDMS) analysis of endogenous cardiac troponin (cTn) complex directly from human heart tissue. The cTn complex is enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complex, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the cTn complex, localizes Ca2+ binding domains, defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a paradigm for structural characterization of endogenous native protein complexes.

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for real-time reaction monitoring to reveal catalytic processes. Here, taking the hydrolase— chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.

Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry.

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

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%.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data. Combining mass determination via spectral deconvolution with a top-down database search and stoichiometry calculations, ProSight Native can determine the complete composition of protein complexes. To demonstrate its features, we used ProSight Native to successfully determine the composition of the homotetrameric membrane complex Aquaporin Z. We also revisited previously published spectra and were able to decipher the composition of a heterodimer complex bound with two noncovalently associated ligands. In addition to determining complex composition, we developed new tools in the software for validating native mass spectrometry fragment ions and mapping top-down fragmentation data onto three-dimensional protein structures. Taken together, ProSight Native will reduce the informatics burden on the growing field of native mass spectrometry, enabling the technology to further its reach.

Native Top-Down Mass Spectrometry Uncovers Two Distinct Binding Motifs of a Functional Neomycin-Sensing Riboswitch Aptamer.

Understanding how ligands bind to ribonucleic acids (RNA) is important for understanding RNA recognition in biological processes and drug development. Here, we have studied neomycin B binding to neomycin-sensing riboswitch aptamer constructs by native top-down mass spectrometry (MS) using electrospray ionization (ESI) and collisionally activated dissociation (CAD). Our MS data for a 27 nt aptamer construct reveal the binding site and ligand interactions, in excellent agreement with the structure derived from nuclear magnetic resonance (NMR) studies. Strikingly, for an extended 40 nt aptamer construct, which represents the sequence with the highest regulatory factor for riboswitch function, we identified two binding motifs for neomycin B binding, one corresponding to the bulge-loop motif of the 27 nt construct and the other one in the minor groove of the lower stem, which according to the MS data are equally populated. By replacing a noncanonical with a canonical base pair in the lower stem of the 40 nt aptamer, we can reduce binding to the minor groove motif from ∼50 to ∼30%. Conversely, the introduction of a CUG/CUG motif in the lower stem shifts the binding equilibrium in favor of minor groove binding. The MS data reveal site-specific and stoichiometry-resolved information on aminoglycoside binding to RNA that is not directly accessible by other methods and underscore the role of noncanonical base pairs in RNA recognition by aminoglycosides.

Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes.

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

Native Top-Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies.

Oligomerization and glycosylation modulate therapeutic glycoprotein stability and efficacy. The interplay between these two critical attributes on therapeutic glycoproteins, is however often hard to define. Here, we present a native top-down mass spectrometry (MS) approach to assess the glycosylation status of therapeutic cytokine and hormone assemblies and relate interfacial glycan occupancy to complex stability. We found that interfacial O-glycan stabilizes tumor necrosis factor-α trimer. On the contrary, interferon-β1a dimerization is independent of glycosylation. Moreover, we discovered a unique distribution of N-glycans on the follicle-stimulating hormone α subunit. We found that the interfacial N-glycan, at Asn52 of the α subunit, interacts extensively with the β subunit to regulate the dimer assembly. Overall, we have exemplified a method to link glycosylation with assembly status, for cytokines and hormones, critical for informing optimal stability and bioavailability.

Native Top-Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies.

Oligomerization and glycosylation modulate therapeutic glycoprotein stability and efficacy. The interplay between these two critical attributes on therapeutic glycoproteins, is however often hard to define. Here, we present a native top-down mass spectrometry (MS) approach to assess the glycosylation status of therapeutic cytokine and hormone assemblies and relate interfacial glycan occupancy to complex stability. We found that interfacial O-glycan stabilizes tumor necrosis factor-α trimer. On the contrary, interferon-β1a dimerization is independent of glycosylation. Moreover, we discovered a unique distribution of N-glycans on the follicle-stimulating hormone α subunit. We found that the interfacial N-glycan, at Asn52 of the α subunit, interacts extensively with the β subunit to regulate the dimer assembly. Overall, we have exemplified a method to link glycosylation with assembly status, for cytokines and hormones, critical for informing optimal stability and bioavailability.

Back Cover: Native Top‐Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies (Angew. Chem. Int. Ed. 49/2022)

Glycan heterogeneity and subunit composition of therapeutic glycoprotein complexes are challenging to define. In their Communication (DOI: 10.1002/anie.202213170), Di Wu and Carol V. Robinson present a native top-down mass spectrometry approach, together with in silico modelling, to reveal the stabilization effect of interfacial N-glycans on therapeutic cytokine and hormone assemblies.

Free Radical-Based Sequencing for Native Top-Down Mass Spectrometry.

Native top-down proteomics allows for both proteoform identification and high-order structure characterization for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmentation methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical-initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS labeling for native top-down sequencing experiments. Our labeling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPO-based FRIPS reagents have the potential to dramatically increase sequence coverage obtained in native top-down experiments.

Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics

The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

On the Mechanism of Theta Capillary Nanoelectrospray Ionization for the Formation of Highly Charged Protein Ions Directly from Native Solutions.

Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to "supercharge" protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS. For θ-nanoESI, dual-channeled nanoESI emitters are used to mix two solutions in low to sub-μs prior to MS. The mechanism for θ-nanoESI mixing has been reported to primarily occur: (i) in a single shared Taylor cone and in the droplets formed from the Taylor cone or (ii) by the fusion of droplets formed from two separate Taylor cones. Using θ-nanoESI-ion mobility MS, native protein solutions were rapidly mixed with denaturing supercharging solutions to form protein ions in significantly higher charge states and with more elongated structures than those formed by premixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone and in the droplets resulting from the single Taylor cone, then the extent of protein charging and unfolding should be comparable to or less than that obtained by premixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. These data also suggest that highly charged protein ions can be formed by the near-complete mixing of each solution. The presence of supercharging additives in premixed solutions can suppress volatile electrolyte evaporation, limiting the extent of protein charging compared to when the additive is delivered via one channel of a θ-nanoESI emitter. In θ-nanoESI, the formation of two Taylor cones can presumably result in substantial electrolyte evaporation from the ESI droplets containing native-like proteins prior to droplet fusion, thereby enhancing ion charging.

Native top-down mass spectrometry for higher-order structural characterization of proteins and complexes.

Progress in structural biology research has led to a high demand for powerful and yet complementary analytical tools for structural characterization of proteins and protein complexes. This demand has significantly increased interest in native mass spectrometry (nMS), particularly native top-down mass spectrometry (nTDMS) in the past decade. This review highlights recent advances in nTDMS for structural research of biological assemblies, with a particular focus on the extra multi-layers of information enabled by TDMS. We include a short introduction of sample preparation and ionization to nMS, tandem fragmentation techniques as well as mass analyzers and software/analysis pipelines used for nTDMS. We highlight unique structural information offered by nTDMS and examples of its broad range of applications in proteins, protein-ligand interactions (metal, cofactor/drug, DNA/RNA, and protein), therapeutic antibodies and antigen-antibody complexes, membrane proteins, macromolecular machineries (ribosome, nucleosome, proteosome, and viruses), to endogenous protein complexes. The challenges, potential, along with perspectives of nTDMS methods for the analysis of proteins and protein assemblies in recombinant and biological samples are discussed.