Protein Higher Order Structure Research Articles

Covalent Labeling Automated Data Analysis Platform for High Throughput in R (coADAPTr): A Proteome-Wide Data Analysis Platform for Covalent Labeling Experiments.

Covalent labeling methods coupled to mass spectrometry have emerged in recent years for studying the higher order structure of proteins. Quantifying the extent of modification of proteins in multiple states (i.e., ligand free vs ligand-bound) can provide information on protein interaction sites and regions of conformational change. Though there are several software platforms that are used to quantify the extent of modification, the process can still be time-consuming, particularly for proteome-wide studies. Here, we present an open-source software for quantitation called Covalent labeling Automated Data Analysis Platform for high Throughput in R (coADAPTr). coADAPTr tackles the need for more efficient data analysis in covalent labeling mass spectrometry for techniques such as hydroxyl radical protein footprinting (HRPF). Traditional methods like Excel's Power Pivot (PP) are cumbersome and time-intensive, posing challenges for large-scale analyses. coADAPTr simplifies analysis by mimicking the functions used in the previous quantitation platform using PowerPivot in Microsoft Excel but with fewer steps, offering proteome-wide insights with enhanced graphical interpretations. Several features have been added to improve the fidelity and throughput compared to those of PowerPivot. These include filters to remove any duplicate data and the use of the arithmetic mean rather than the geometric mean for quantitation of the extent of modification. Validation studies confirm coADAPTr's accuracy and efficiency while processing data up to 200 times faster than conventional methods. Its open-source design and user-friendly interface make it accessible for researchers exploring intricate biological phenomena via HRPF and other covalent labeling MS methods. coADAPTr marks a significant leap in structural proteomics, providing a versatile and efficient platform for data interpretation. Its potential to transform the field lies in its seamless handling of proteome-wide data analyses, empowering researchers with a robust tool for deciphering complex structural biology data.

Radical Protein Footprinting in Mammalian Whole Blood.

Hydroxyl Radical Protein Footprinting (HRPF) is a powerful method to probe the solvent-accessible surface area of proteins. It is mostly used to study the higher-order structure of proteins, as well as protein-protein and protein-carbohydrate interactions. Hydroxyl radicals are generated by the photolysis of hydrogen peroxide and these radicals modify the surface amino acids. Bottom-up proteomics is then applied and peptide oxidation is calculated and correlated with solvent accessibility. It is mainly performed in vitro ; however, it has been recently used in living systems, including live cells, live nematodes, and 3D cell cultures. Mammalian tissues are still out of reach as they absorb UV strongly, hindering radical generation. Here, we describe the first example of RPF in mammalian stabilized whole blood. Using photoactivation of persulfate with a commercially available FOX Photolysis System modified for sample handling and inline mixing, we demonstrate the first labeling of proteins in whole blood. We demonstrate that the RPF protocol does not alter the blood cell gross morphology outside of a moderate hypertonicity equivalent to sodium chloride exposure prior to labeling. We detail an improved quenching protocol to limit background labeling in persulfate RPF. We describe the labeling of the top ten most abundant proteins in the blood. We demonstrate the equivalence of ex vivo labeling in whole blood with labeling of the same structure in vitro using hemoglobin as a test system. Overall, these results now open the possibility of performing RPF-based structural proteomics in pre-clinical models and using readily available clinical samples.

Interrogation of three-finger toxin and phospholipase A2 higher order structures from the forest cobra (Naja melanoleuca) venom using a mass spectrometric approach

Snake venoms are composed of bioactive proteins and peptides from various toxin families and elicit potent pharmacological activity. There is great interest in characterising these venom proteins for the development of effective antivenom treatment as well as utilisation for biomedical and therapeutic applications. However, a thorough structural understanding of the snake venom proteins is necessary. Higher-order protein complexes are known to form in snake venoms and lend structural and functional diversity, often eliciting greater activity than the sum of monomeric protein species. Despite the significance, the nature of these protein complexes is extremely underexplored. In this study, we demonstrate the use of mass spectrometry (MS)-based strategies to explore the toxins at a quaternary level in the venom from the medically significant forest cobra (Naja melanoleuca). Small toxins, mainly three finger toxins (3FTxs) and phospholipase A2s (PLA2s), were identified by comparison of intact and chemically reduced masses using matrix-assisted laser desorption ionisation (MALDI-MS) profiling. Notably, interrogation of these small toxins by native MS and collision-induced dissociation revealed the presence of various non-covalent 3FTx and PLA2 dimers, providing insight on the higher-order protein structures for a variety of N. melanoleuca toxins using a MS-based approach. Furthermore, phospholipid substrate specificity of N. melanoleuca PLA2 enzymes were explored, capturing the indiscriminate activity of these PLA2s towards a range of phospholipid classes for the first time.

Martini 3 OliGo̅mers: A Scalable Approach for Multimers and Fibrils in GROMACS.

Martini 3 is a widely used coarse-grained simulation method for large-scale biomolecular simulations. It can be combined with a Go̅ model to realistically describe higher-order protein structures while allowing the folding and unfolding events. However, as of today, this method has largely been used only for individual monomers. In this article, we describe how the Go̅ model can be implemented within the framework of Martini 3 for a multimer system, taking into account both intramolecular and intermolecular interactions in an oligomeric protein system. We demonstrate the method by showing how it can be applied to both structural stability maintenance and assembly/disassembly of protein oligomers, using aquaporin tetramer, insulin dimer, and amyloid-β fibril as examples. We find that addition of intermolecular Go̅ potentials stabilizes the quaternary structure of proteins. The strength of the Go̅ potentials can be tuned so that the internal fluctuations of proteins match the behavior of atomistic simulation models, however, the results also show that the use of too strong intermolecular Go̅ potentials weakens the chemical specificity of oligomerization. The Martini-Go̅ model presented here enables the use of Go̅ potentials in oligomeric molecular systems in a computationally efficient and parallelizable manner, especially in the case of homopolymers, where the number of identical protein monomers is high. This paves the way for coarse-grained simulations of large protein complexes, such as viral protein capsids and prion fibrils, in complex biological environments.

Therapeutic Application and Structural Features of Adeno-Associated Virus Vector.

Adeno-associated virus (AAV) is characterized by non-pathogenicity, long-term infection, and broad tropism and is actively developed as a vector virus for gene therapy products. AAV is classified into more than 100 serotypes based on differences in the amino acid sequence of the capsid protein. Endocytosis involves the uptake of viral particles by AAV and accessory receptors during AAV infection. After entry into the cell, they are transported to the nucleus through the nuclear pore complex. AAVs mainly use proteoglycans as receptors to enter cells, but the types of sugar chains in proteoglycans that have binding ability are different. Therefore, it is necessary to properly evaluate the primary structure of receptor proteins, such as amino acid sequences and post-translational modifications, including glycosylation, and the higher-order structure of proteins, such as the folding of the entire capsid structure and the three-dimensional (3D) structure of functional domains, to ensure the efficacy and safety of biopharmaceuticals. To further enhance safety, it is necessary to further improve the efficiency of gene transfer into target cells, reduce the amount of vector administered, and prevent infection of non-target cells.

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.