Protein Higher Order Structure Research Articles

Biosensing-based quality control monitoring of the higher-order structures of therapeutic antibody domains

Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.

Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles.

Collision induced unfolding (CIU) is a method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the gas-phase unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation, together with CIU experiments. The dimers have highly similar structures, but their CIU reveals different stability that can be explained by the different dynamics that arises in response to the activation seen in the simulations, including a part of the sequence with previously observed strain-specific dynamics in solution. Our findings show how similar protein variants can be examined using mass spectrometric techniques in conjunction with atomistic molecular dynamics simulations to reveal differences in stability as well as differences in how and where unfolding takes place upon activation.

Online Coupling of Size Exclusion Chromatography to Capillary Enhanced Raman Spectroscopy for the Analysis of Proteins and Biopharmaceutical Drug Products.

The online coupling of size exclusion chromatography (SEC) to capillary enhanced Raman spectroscopy (CERS) based on a liquid core waveguide (LCW) flow cell was applied for the first time to assess the higher-order structure of different proteins. This setup allows recording of Raman spectra of the monomeric protein within complex mixtures, since SEC enables the separation of the monomeric protein from matrix components such as excipients of a biopharmaceutical product and higher molecular weight species (e.g., aggregates). The acquired Raman spectra were used for structural elucidation of well characterized proteins such as bovine serum albumin, hen egg white lysozyme, and β-lactoglobulin and of the monoclonal antibody rituximab in a medicinal product. Additionally, the CERS detection of the disaccharide sucrose, which is used as a stabilizing excipient, was quantified to achieve a limit of detection (LOD) of 120 μg and a limit of quantification (LOQ) of 363 μg injected on the column.

Protein-Based Patterning to Spatially Functionalize Biomimetic Membranes.

The bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.

Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Combined Liquid-State and Solid-State Nuclear Magnetic Resonance at Natural Abundance for Comparative Higher Order Structure Assessment in the Formulated-State of Biphasic Biopharmaceutics.

A higher-order structure (HOS) is critical to a biopharmaceutical drug as the three-dimensional structure governs its function. Even the partial perturbation in the HOS of the drug can alter the biological efficiency and efficacy. Due to current limitations in analytical technologies, it is imperative to develop a protocol to characterize the HOS of biopharmaceuticals in the native formulated state. This becomes even more challenging for the suspension formulations where solution and solid phases co-exist. Here, we have used a combinatorial approach using liquid (1D 1H) and solid-state (13C CP MAS) NMR methodology to demonstrate the HOS in the biphasic microcrystalline suspension drug in its formulated state. The data were further assessed by principal component analysis and Mahalanobis distance (DM) calculation for quantitative assessment. This approach is sufficient to provide information regarding the protein HOS and the local dynamics of the molecule when combined with orthogonal techniques such as X-ray scattering. Our method can be an elegant tool to investigate batch-to-batch variation in the process of manufacture and storage as well as a biosimilarity comparison study for biphasic/microcrystalline suspension.

Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder

BackgroundSpray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions.MethodsVisible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX).ResultsParticles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying.DiscussionThe spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.

Advances in Mass Spectrometry on Membrane Proteins.

Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins' dynamic nature and heterogeneity. Mass spectrometry (MS) is emerging as a powerful tool for investigating membrane protein structure and dynamics. Studying MPs using MS, however, must meet several challenges including the lack of stability and solubility of MPs, the complexity of the protein-membrane system, and the difficulty of digestion and detection. To meet these challenges, recent advances in MS have engendered opportunities in resolving the dynamics and structures of MP. This article reviews achievements over the past few years that enable the study of MPs by MS. We first introduce recent advances in hydrogen deuterium exchange and native mass spectrometry for MPs and then focus on those footprinting methods that report on protein structure.

Diverse Fluorescence Resonance Energy Transfer Processes Originating from the Conformational Heterogeneity of the Calcium Indicator Yellow Cameleon YC3.60.

By combining time-resolved fluorescence spectroscopy and molecular biology, we have clearly elucidated the fluorescence mechanism of yellow cameleon YC3.60. YC3.60 is one of the most widely used fundamental calcium ion indicator proteins and is a tandem protein consisting of cyan fluorescent protein, calmodulin, and yellow fluorescent protein. Our results show that the conformational heterogeneity of YC3.60 leads to multiple FRET (fluorescence resonance energy transfer) processes occurring in a variety of structures. In the presence of calcium ions, FRET occurs in 75% of all YC3.60 molecules, and the intramolecular FRET faster than 20 ps is dominant among the three different FRET processes. Even in the absence of calcium ions, FRET occurs in 28% of YC3.60, where intra- and intermolecular FRET with a time constant of 160 ps is dominant among the four different FRET processes. YC3.60 with the immature chromophore, which is 25% of the total, would result in lower contrast on imaging. Conformational heterogeneity arises from the specific association of the two fluorescent proteins. It was clearly shown that the association of fluorescent proteins has a significant impact on the fluorescence mechanism. These observations lead to the promotion of research that elucidates the relationship between the higher-order structure and function of proteins.

Direct Assessment of Oligomerization of Chemically Modified Peptides and Proteins in Formulations using DLS and DOSY-NMR.

Protein higher order structure (HOS) including the oligomer distribution can be critical for efficacy, safety and stability of drug products (DP). Oligomerization is particularly relevant to chemically modified protein therapeutics that have an extended pharmacokinetics profile. Therefore, the direct assessment of protein oligomerization in drug formulation is desired for quality assurance and control. Here, two non-invasive methods, dynamic light scattering (DLS) and diffusion ordered spectroscopy (DOSY) NMR, were applied to measure translational diffusion coefficients (Ddls and Dnmr) of proteins in formulated drug products. The hydrodynamic molecular weights (MWhd), similar to hydrodynamic size, of protein therapeutics were derived based on a log(Ddls) vs log(MWhd) correlation model established using protein standards. An exponent value of -0.40 ± 0.01 was established for DLS measured log(D) vs. log(MWhd) using protein standards and a theoretical exponent value of -0.6 was used for unstructured polyethylene glycol (PEG) chains. The analysis of DLS derived MWhd of the primary species showed the fatty acid linked glucagon-like peptide 1 (GLP-1) was in different oligomer states, but the fatty acid linked insulin and PEG linked proteins were in monomer states. Nevertheless, equilibrium and exchange between oligomers in formulations were universal and clearly evidenced from DOSY-NMR for all drugs except peginterferon alfa-2a. The correlation models of log(D) vs. log(MWhd) could be a quick and efficient way to predict MWhd of protein, which directly informs on the state of protein folding and oligomerization in formulation.

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.

Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material.

Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L-1 (k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433].

Hydroxyl radical protein footprinting for analysis of higher order structure

Tabled 1aAdapted from Johnson et al. [11].Hydrogen deuterium exchangeGlycine ethyl esterDiethylpyrocarbonateN-Ethylmaleimide (NEM)Fast photochemical oxidation of proteinsLabeling timescaleSeconds to hoursSeconds to minutesMinutesMinutes to hoursMicrosecondsType of labelReversibleReversibleReversible for most residuesIrreversible for LysIrreversibleIrreversibleSensitivity/specificityBackbone amide groupsCarboxylic acidsNucleophilic residuesN terminus, His, Lys, Tyr, Ser, Thr, and CysCys19 out of 20 amino acids (excluding Gly)Chemicals/materialsCommonly used buffers under physiological conditionsQuench solutionspH 2.5 (strong or weak acid)0°CDenaturing (urea,guanidine HCl)Reducing agents (TCEP)Pepsin digestionEDC stock solutionprepared freshly in PBS bufferMES/NaOH (pH = 5.5),PBS (pH = 6, 6.5, 7, 7.5),HEPES (pH = 8, 8.5)DEPC in acetonitrile in a molar excess of 4 10 mM imidazole added to quench the reactionNEM solution10 mm HEPES5% DMSOVarious buffer solutions based on proteinH2O2free glutamine radical scavengerQuench solution: free methionine and catalase (in vitro)LimitationsReversibility of the label limits its use for complex samples that require longer separation timesCarboxylic acid-specific labeling limits the proteins that can be studiedFree cysteine scramblingCysteine-specific labeling limits the proteins that can be studiedResidue-reactivity differences, complex data analysis, and expense of excimer lasera Adapted from Johnson et al. [11]. Open table in a new tab Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems.ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput.CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems.Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. Hydroxyl radicals can be generated using various methods, including Fenton chemistry, radiolysis of water, and photolysis of hydrogen peroxide. The labeling timescale is based on the radical generation method with some labeling proteins on the nano- or microsecond timescale, which is faster than all other footprinting methods. HRPF has been successful in studying HOS in varied applications, including epitope mapping, ion channel dynamics, and viral protein studies. HRPF has also been used to study intact cells and in vivo systems. ADVANTAGES:General labeling strategy that can modify 19 of the 20 amino acids.Labeling is compatible with several widely used buffer systems.The irreversible nature of the label provides flexibility for postlabeling sample handling.Can identify protein interaction sites and regions of conformational changes.Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans.Advancements in dosimetry have increased the quantitative power of the method.There have been efforts to automate labeling leading to higher throughput. General labeling strategy that can modify 19 of the 20 amino acids. Labeling is compatible with several widely used buffer systems. The irreversible nature of the label provides flexibility for postlabeling sample handling. Can identify protein interaction sites and regions of conformational changes. Can analyze complex systems such as cell lysates, intact cells, and Caenorhabditis elegans. Advancements in dosimetry have increased the quantitative power of the method. There have been efforts to automate labeling leading to higher throughput. CHALLENGES:Data analysis is complex, time consuming, and there is not a universal data analysis platform.Generating hydroxyl radicals on a fast timescale requires special instrumentation.Lower resolution than other structural methods.Difficult to identify interacting proteins in complex systems. Data analysis is complex, time consuming, and there is not a universal data analysis platform. Generating hydroxyl radicals on a fast timescale requires special instrumentation. Lower resolution than other structural methods. Difficult to identify interacting proteins in complex systems. Declaration of interestsL.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. L.M.J. discloses a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis.

Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry.

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

Identifying glycation hot-spots in bovine milk proteins during production and storage of skim milk powder

We investigated protein glycation in a complex milk system under controlled conditions representative of real-life consumer products, analysing intermediate and final products from skim milk powder production, and aged powder samples. We combined protein-centric LC-MS(/MS) with peptide-centric multi-protease LC-MS/MS focusing on the six most abundant bovine milk proteins. This strategy resulted in the identification of glycated proteoforms and of the extent of glycation per protein, high protein sequence coverage, and identification and relative occupancy of the glycation sites. We identified new glycation hot-spots additionally to the ones already described in literature. Primary sequence motif analysis revealed that glycation hot-spots were preceded N-terminally by a stretch rich in basic amino acids, and followed C-terminally by a stretch enriched in aliphatic and hydrophobic amino acids. Our study considerably extends the current understanding of milk protein glycation, discussing glycation hot-spots and their localisation in relation to the primary sequences and higher-order protein structures.

Studying protein structure and function by native separation-mass spectrometry.

Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure-function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions.

Enhanced rehydration behaviors of micellar casein powder: The effects of high hydrostatic pressure treatments on micelle structures

Natural micellar casein is generally dried into powdered forms for commercial transportation and storage. However, the poor rehydration ability of micellar casein powder critically limited the potential applications due to its dense cross-linked structures caused by colloidal calcium phosphate (CCP). In this study, micellar casein solutions were exposed to a high hydrostatic pressure (HHP) ranging from 100 to 500 MPa and were then freeze dried to produce powders. The effects on the casein micelle structures and the rehydration characteristics including wetting, dispersion and dissolving were comprehensively investigated. The results showed that HHP could induce smaller micelle sizes and significantly increase the free calcium in the reconstituted solution. It demonstrated that the majority of CCP bridges in casein micelles were dissociated, which produced porous powders with loose structures and thus significantly improved rehydration behaviors. 300 MPa was the pressure level that caused the quickest dispersion process and best solubility. Consequently, HHP has potential to be a novel physical technique to potentially modify the protein higher-order structures as well as improve the corresponding functionalities.

Exploring the Potential of Molecular Spectroscopy for the Detection of Post-translational Modifications of a Stressed Biopharmaceutical Protein.

Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex arrangements (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as posttranslational modifications (PTMs). PTMs may affect the protein's functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins. In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs. This work required some novel computational approaches to pre-process Raman and IR spectra and a review of the band assignments for proteins existing in the literature.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins.

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method, which utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids, can inform on protein interaction sites and regions of conformational change. Hydroxyl radical-based footprinting was originally developed to study nucleic acids, but coupling the method with mass spectrometry has enabled the study of proteins. The method has undergone several advancements since its inception that have increased its utility for more varied applications such as protein folding and the study of biotherapeutics. In addition, recent innovations have led to the study of increasingly complex systems including cell lysates and intact cells. Technological advances have also increased throughput and allowed for better control of experimental conditions. In this review, we provide a brief history of the field of HRPF and detail recent innovations and applications in the field.