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Schematic representation of hydroxyl radical footprinting of proteins coupled with mass spectrometry. Proteins are oxidatively modified by hydroxyl radicals that can be generated by a variety of methods. Modified proteins are digested and analyzed by LC-MS/ MS.
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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...
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... These methods utilize covalent labels to modify proteins, and these labels are identified and quantified using high-resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS). A subset of these methods is hydroxyl radical protein footprinting (HRPF) which utilizes hydroxyl radicals to oxidatively modify the side chains of amino acids ( Figure 1). Hydroxyl radical-based footprinting has been used for decades and was originally developed to study nucleic acids. ...
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... these, all but two, AZ and OA, did not scavenge the radical, so these two were further tested to determine whether they increased H 2 O 2 uptake. The fluorescence of the fluorophore, PO1, which has been used to determine the in vivo hydrogen peroxide uptake and diffusion in C. elegans, was used to demonstrate the increased hydrogen peroxide uptake that occurred after incubation with AZ or OA ( Figure 10A−C). Noteworthy, only AZ at 1% provided a statistical difference in increasing hydrogen peroxide uptake with a 1.24-fold increase ( Figure 10D). ...
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... fluorescence of the fluorophore, PO1, which has been used to determine the in vivo hydrogen peroxide uptake and diffusion in C. elegans, was used to demonstrate the increased hydrogen peroxide uptake that occurred after incubation with AZ or OA ( Figure 10A−C). Noteworthy, only AZ at 1% provided a statistical difference in increasing hydrogen peroxide uptake with a 1.24-fold increase ( Figure 10D). The increased uptake of hydrogen peroxide was also reflected in the number of proteins oxidatively modified by IV-FPOP with 1084 and 1140 for normal IV-FPOP and IV-FPOP + azone, respectively. ...
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... experiments, consisting of three biological replicates, without (label free) and with cPILOT multiplexing were compared. The three biological replicates that were not multiplexed had only 48 proteins in common ( Figure 11A), whereas the three biological replicates that underwent cPILOT had 429 mutual proteins, an approximately 9-fold increase ( Figure 11B). In addition to its utility at the global level, cPILOT also improves reproducibility at the peptide level, providing greater confidence in localizing modifications. ...
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... experiments, consisting of three biological replicates, without (label free) and with cPILOT multiplexing were compared. The three biological replicates that were not multiplexed had only 48 proteins in common ( Figure 11A), whereas the three biological replicates that underwent cPILOT had 429 mutual proteins, an approximately 9-fold increase ( Figure 11B). In addition to its utility at the global level, cPILOT also improves reproducibility at the peptide level, providing greater confidence in localizing modifications. ...
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... addition to its utility at the global level, cPILOT also improves reproducibility at the peptide level, providing greater confidence in localizing modifications. In cPILOT-free experiments, 11 modified peptides were observed for the protein tubulin beta chain, none of which were mutually observed across the biological replicates ( Figure 11C). However, in the cPILOT replicates six modified peptides observed for tubulin, four of which were found in all three biological replicates ( Figure 11D). ...
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... cPILOT-free experiments, 11 modified peptides were observed for the protein tubulin beta chain, none of which were mutually observed across the biological replicates ( Figure 11C). However, in the cPILOT replicates six modified peptides observed for tubulin, four of which were found in all three biological replicates ( Figure 11D). Although a higher number of peptides across the protein were quantified for the label free studies, more peptides were reproducibly quantified across biological replications, providing better statistical significance. ...
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... overcome this, Riaz et al. developed a static platform where laser photolysis was performed in a 96-well microtiter plate. 111 The laser beam was deflected downward onto the plate using a laser line mirror ( Figure 12A). The the sample was exposed to a single pulse of the laser beam to avoid oxidation-induced unfolding. ...
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... using a V-shaped well plate, to overcome UV shadowing of the sample near the well wall, adenine response to increased laser fluence was linear, comparable to traditional FPOP with the flow system. A similarity in oxidation between microtiter and traditional FPOP was also observed for the model peptide GluB ( Figure 12B). A comparison of the extent of FPOP modification on myoglobin showed similar levels of labeling, demonstrating the equivalence of the two methods ( Figure 12C). ...
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... similarity in oxidation between microtiter and traditional FPOP was also observed for the model peptide GluB ( Figure 12B). A comparison of the extent of FPOP modification on myoglobin showed similar levels of labeling, demonstrating the equivalence of the two methods ( Figure 12C). This comparable level of labeling was observed even though the flow system method utilizes a 15% exclusion volume to limit exposure of the sample to multiple laser pulses. ...
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... high-throughput static platform was also developed for IC-FPOP by Johnson et al. 112 This platform, called platform incubator with XY movement (PIXY), consists of a stage-top incubator, movable stage, and peristaltic pumps ( Figure 13A− D). The laser beam is deflected down onto the incubator using 50 mm laser mirrors like with the study by Riaz et al. 111 PIXY takes advantage of the fact that mammalian cells can be cultured in adhered monolayers in culture plates. ...
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... information was obtained from the titration of a high concentration of protein (10 μM), which is 3000-fold greater than the K d of the system, using global-level data that was acquired using the intact protein. This high-concentration titration led to a sharp break curve that revealed that the binding stoichiometry was 1:1 ( Figure 14A). A smooth decay is observed for a lowconcentration titration (holo-CaM = 200 nM) ( Figure 14B). ...
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... high-concentration titration led to a sharp break curve that revealed that the binding stoichiometry was 1:1 ( Figure 14A). A smooth decay is observed for a lowconcentration titration (holo-CaM = 200 nM) ( Figure 14B). Peptide-level data from the proteolyzed protein was obtained using a lower protein concentration (200 nm) and informed on binding sites and site-specific binding affinity. ...
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... this work, apo-calmodulin was titrated with Ca 2+ ions followed by FPOP and proteolysis, which allowed the binding behavior to be evaluated section by section. The results of the experiments revealed 4 major classes: Class I, which undergo a loss of protection, Class II, which undergo no change in protection, Class III, which become protected, and Class IV, which capture the allosteric effects of calcium Figure 14. Modification fraction measured at the global level as a function of melittin concentration with initial protein concentrations of (A) 10 μM (high-concentration titration exhibits a sharp break curve that indicates the binding stoichiometry is 1:1) and (B) 200 nM (low-concentration titration results in a curve with a smooth decay). ...
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... To select relevant mutants, Ashcroft drew on findings from mutational analysis which determines the significance of a mutation in stabilizing or destabilizing folding. 141 To have representation from different points along the folding pathway, two mutants were selected: DM Im7, which retains native-like structural properties, and TM Im7, which contains mutations that stabilize the unfolded state ( Figure 15A). DM IM7 has two mutations and retains nativelike secondary structure in helices I and IV, partial formation of helix II, and complete loss of helix III. ...
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... native electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) did not distinguish between wild type and the two mutants, with each displaying similar collisional cross sections even though TM IM7 has a larger hydrodynamic radius in solution. In contrast, global FPOP oxidation showed a clear difference in HOS with both mutants having a higher extent of modification than wild type, presumably due to alterations in the secondary structure ( Figure 15B). Peptidelevel analysis localized these structural differences. ...
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... analysis localized these structural differences. Although there was an overall increase in oxidation for DM IM7 compared to wild type, this increase was only observed in two of the three peptides, 5−20 and 44−70, and modified in this mutant ( Figure 15C). Peptide 44−70 corresponds to helix III of the protein that is known to be unfolded in DM IM. ...
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... The results of this endeavor demonstrated the utility of FPOP and HDX as each technique had regions that were unique to the other. HDX was able to inform on regions that were unreactive to hydroxyl radicals and thus did not produce oxidation products (i.e., peptide 143−153), and FPOP, which labels on a faster time scale, can detect changes that occur rapidly (i.e., peptide 124−139) ( Figure 16B). After localizing regions that participate in epitope binding ( Figure 16A) via the footprinting experiments, computational epitope predictions were used to propose relevant mutants and alanine shave mutagenesis was used to determine the energetic determinants. ...
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... was able to inform on regions that were unreactive to hydroxyl radicals and thus did not produce oxidation products (i.e., peptide 143−153), and FPOP, which labels on a faster time scale, can detect changes that occur rapidly (i.e., peptide 124−139) ( Figure 16B). After localizing regions that participate in epitope binding ( Figure 16A) via the footprinting experiments, computational epitope predictions were used to propose relevant mutants and alanine shave mutagenesis was used to determine the energetic determinants. The researchers point out that their method, which determines residues responsible for epitope binding, allows optimization in developing novel therapeutics. ...
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... residues were located in the same regions as the shorter incubation time HDX data, demonstrating the complementarity of these methods. Interestingly, for several modified residues, multiple peaks at different retention times were observed in the extracted ion chromatogram for the same peptide ( Figure 17). ...
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... were attributed to the presence of isomers where a single residue is modified at different sites. Phe, which can be oxidized at its ortho, meta, and para positions consistently showed three retention time peaks for Phe22, Phe30, and Phe70 ( Figure 17). The three modified tyrosines each had two peaks consistent with ortho and meta isomers. ...
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... Size-exclusion chromatography/multiangle light scattering (SEC/MALS) and native MS were used to examine the protein complex formation and binding specificity of the binary complexes (BsAb1/CD3 and BsAb1/BCMA) and the ternary complex, BsAb1/CD3/BCMA. Subsequently, the trio of footprinting techniques was used to resolve the location of the epitopes as a binary complex and a ternary complex (Figure 18). The authors acknowledge this is the first demonstration of an integrated approach for epitope mapping of a BsAb in both binary and ternary complexes. ...
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... Previous work suggested that the conformational transition drives the aggregated protein through the ring of the hexamer where the protein can be disaggregated. 183,184 By coupling the sensitivity to conformational change of HRPF to the previous cryo-EM studies, Sweeny and Shorter identified critical regions/residues needed for optimal Hsp104 function (Figure 19). To do this, the investigators performed HRPF on Hsp104 in three conditions: (i) Hsp104 monomers in the absence of nucleotide, (ii) Hsp104 hexamers in the presence of ADP, and (iii) Hsp104 hexamers in the presence of ATPγS. ...
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... where amino acid hydroxylation induced by water ionization through x-ray exposure can be measured by mass spectrometry, 50,51 to provide experimental support for these findings? ...
Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria which encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semi-permeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles, typically trimers, hexamers, or pentamers, and these tiles assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We use molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.
... To understand the lower relative degree of oxidation products for longer peptide chains, we compare our results to "Fast Photochemical Oxidation of Protein" (FPOP), which is a method in mass spectrometry that generates OH • radicals in solution with µs lifetimes to label native proteins. 34,35 In FPOP experiments, only 20-60% of protein may remain unmodified. 36 The native conformation is generally retained because the reaction of the OH • radicals is faster than the conformational dynamics of the average protein. ...
Low-temperature plasma (LTPs) jets are FDA-approved medical devices to remove cancerous tissue and aid in wound healing. However, reports on their reaction with proteins are conflicting, ranging from fragmentation, oxidation, aggregation, or a combination thereof. In this study we bridge the gap between plasma-treatment of short peptides to proteins at physiologically relevant concentrations. The LTP in this study is based on a helium dielectric barrier discharge (DBD) that forms a plasma-jet, which is directed at the solution without direct contact with the plasma, and results in the formation of reactive oxygen species (ROS) OH• and O2•- in solution. The longer the solution is treated, the more solution-phase ROS form. Treating peptide- and protein-containing solutions leads to extensive oxidation. The ROS led to the same oxidative modifications for peptide M with increasing chain length (9, 18, 37, 76 amino acids), which could be identified with high-resolution mass spectrometry. Oxidized species M+xO led to conformational changes such as compaction and elongation, while the unmodified peptide M remained unaltered, as found by ion mobility spectrometry and size exclusion chromatography. For proteins at high concentration, insoluble aggregates formed and could be identified by UV/Vis light scattering and atomic force microscopy. The formation of aggregates is dependent on the amino acid chain length, the peptide concentration, and the time for aggregate formation. These findings highlight the importance of both peptide chain length and concentration in determining the fate of peptides following the exposure to LTP, while also offering valuable insights for the field of plasma medicine.
... One approach, hydroxyl radical protein footprinting (HRPF) has been demonstrated in IDP characterization (20); HRFP was shown to detail changes in protein modification as a function of Aβ1-42 aggregation (20). The use of HRFP for characterizing IDPs is especially intriguing considering the recent extension of the labeling approach to in-cell analysis (21). ...
... One approach, hydroxyl radical protein footprinting (HRPF) has been demonstrated in IDP characterization (20); HRFP was shown to detail changes in protein modification as a function of Aβ1-42 aggregation (20). The use of HRFP for characterizing IDPs is especially intriguing considering the recent extension of the labeling approach to in-cell analysis (21). ...
Behaving allosterically in nature and possessing versatile structural conformational types, intrinsically disordered proteins (IDPs) as well as intrinsically disordered regions (proteins) exhibit multi-functional properties in living organisms. However, their dynamic nature composed of conformational fluctuations over very brief timescales has made characterizing solution states challenging. In this research, for the first time, a method to facilitate the conformational mapping of challenging peptide systems has been derived from in-droplet hydrogen/deuterium exchange and mass spectrometry (HDX-MS) data combined with molecular dynamics (MD) simulations. Field-enabled capillary vibrating sharp-edge sharp spray ionization (cVSSI) devices are coupled to MS to measure HDX reactions for structurally varied peptide systems including: polyalanine (PA), bradykinin (BK), model peptide (KDD), polyserine (PS), and the first 17 amino acid residues of the Exon 1 region of Huntingtin protein (Nt17) in the presence of internal standard (e.g., lysine and arginine). Initially, we determined that propensity factor of different heteroatoms hydrogen which characterized the hydrogen type in peptide of interests. Here, we have reported 86% deuterium incorporation of lysine in electro-osmotically favored mixing of analyte and D2O droplet in-droplet HDX measurements. From the estimated backbone amide deuterium incorporation data, MD simulations were harnessed to interpret and predict HDX behavior for peptides in a systematic fashion. Initially, a sequence-based intrinsic rate approach, which takes into account steric and electronic effects on backbone amide sites, showed a poor ability (correlation coefficient of 0.27) to predict backbone deuterium incorporation. A second approach which included the consideration of secondary and tertiary structure protection as well as the ability to form intra- and inter-molecular hydrogen bonds provided improved reaction prediction capability (correlation coefficient values of 0.79 and 0.90, respectively). Further modifying the model by including scaled relative intrinsic rates significantly improves reaction predictability (correlation coefficient values of 0.97 and 0.99, respectively). The newly developed model provides a crucial link between experimental and theoretical realms allowing interpretation of HDX-MS data. Remarkably, the novel approach suggests a strong protective role for structural rigidity along with secondary structure protection accounting for decreased deuterium incorporation especially for BK. Furthermore, application of the model to the Nt17 system suggests that, although the peptide may be considered as intrinsically disordered in nature, the high frequency formation of very transient secondary structure across the peptide sequence (overall higher inter-residue contact) render it relatively inflexible to HDX. That said, such structural fluctuation may also provide clues about the ability of the amphipathic Nt17 peptide to rapidly stabilize helical structure upon interaction with binding partners (lipid and/or peptide) and thus participate in aggregation phenomena associated with Huntington’s Disease (HD). The results lay the groundwork for further improvements of the model and its potential application in the study of IDP and IDR structural properties for many important proteins.
... Thus, identifying and quantifying peptides bearing covalently labeled residues via high-throughput proteomics analysis has become a stalwart in the arsenal of structural mass spectrometry (MS) tools to uncover l ig an d-bi nding sites and to profile binding-induced c o n fo r m at ional c h a n g e s 24 . A mo ng t h e v e r sa tile l a b el ing m e t ho ds, exemplified by amino acid-specific labeling 24 and indiscriminate hydroxyl radical footprinting 25 , lysine dimethylation is highly efficient and has been successfully used to profile changes in lysine accessibility due to ligand engagement for purified proteins 26 ; however, the method is rarely used for biologically complex systems. Recently, this method was extended to recognize protein misfolding in complex brain lysates and living cells by covalent protein painting 27 . ...
... This explains why the responsive accessibilities of these bona fide targets were not captured by TRAP and penalized righteous assignment. Future expansion to versatile labeling methods, such as those targeting amino acid residues 24 present in the ligand-binding domain at high frequency (histidine, tryptophan and tyrosine) 53 , and residue-indiscriminate methods, such as footprinting 25,54 and hydrogen-deuterium exchange 55 , may substantially broaden the accessible landscape. Furthermore, some cases occurred when we successfully identified labeled lysines in metabolite-interacting domains from known target proteins; however, no responsive accessibility was detected. ...
Hyperactivated glycolysis is a metabolic hallmark of most cancer cells. Although sporadic information has revealed that glycolytic metabolites possess nonmetabolic functions as signaling molecules, how these metabolites interact with and functionally regulate their binding targets remains largely elusive. Here, we introduce a target-responsive accessibility profiling (TRAP) approach that measures changes in ligand binding-induced accessibility for target identification by globally labeling reactive proteinaceous lysines. With TRAP, we mapped 913 responsive target candidates and 2,487 interactions for 10 major glycolytic metabolites in a model cancer cell line. The wide targetome depicted by TRAP unveils diverse regulatory modalities of glycolytic metabolites, and these modalities involve direct perturbation of enzymes in carbohydrate metabolism, intervention of an orphan transcriptional protein’s activity and modulation of targetome-level acetylation. These results further our knowledge of how glycolysis orchestrates signaling pathways in cancer cells to support their survival, and inspire exploitation of the glycolytic targetome for cancer therapy.
... DE peptide abundance levels reflect only conformational differences localized to the DE peptide itself and its linked residue. In fact, DE peptides provide information analogous to other covalent labeling techniques such as radical footprinting 22 or HDX 23 , albeit with less depth, precision, or temporal dynamics than either of these methods. Here, we describe how a direct comparison between the relative abundance of cross-linked peptide pairs and their DE peptide counterparts in samples derived from animals with phenotypic differences can help elucidate the conformational causes of observed cross-link abundance differences, whether they involve an alteration in the solvent-accessible distance between the cross-linked residues or changes of other conformational features localized to one or both residues. ...
XL-MS provides low-resolution structural information of proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells, or young and old mice. A difference can originate from protein conformational changes altering the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications on the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein, the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences of cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.
... Advances in mass spectrometry (MS) have addressed some of these shortcomings through significantly improved throughput and accessibility, and facile integration with chemical biology tools has expanded its applications. Chemical crosslinking, footprinting, limited proteolysis, hydrogen-deuterium exchange, and other related methods that were once almost exclusively used for purified protein samples have been successfully applied to studies of protein structure and function at the proteome scale (Chavez et al., 2022;James et al., 2022;Mateus et al., 2016;McKenzie-Coe et al., 2022;Petrotchenko & Borchers, 2022;Piersimoni et al., 2022;Schopper et al., 2017;Wheat et al., 2021). These "structural proteomics" methods introduce structure-dependent changes in proteins in their native states (e.g., via chemical labeling or proteolysis) that can then be measured by MS (Figure 1). ...
A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.
... S. Li et al., 2018;X. R. Liu et al., 2020;McKenzie-Coe et al., 2022) provides increased structural information on proteins by inducing promiscuous oxidation of various residues. However, when used in the cellular context, the proteomics analysis of individual oxidized peptides can be challenging, especially those of lowabundance proteins. ...
Chemical proteomics, which involves studying the covalent modifications of proteins by small molecules, has significantly contributed to our understanding of protein function and has become an essential tool in drug discovery. Mass spectrometry (MS) is the primary method for identifying and quantifying protein-small molecule adducts. In this review, we discuss various methods for measuring proteomic reactivity using MS and covalent proteomics probes that engage through reactivity-driven and proximity-driven mechanisms. We highlight the applications of these methods and probes in live-cell measurements, drug target identification and validation, and characterizing protein-small molecule interactions. We conclude the review with current developments and future opportunities in the field, providing our perspectives on analytical considerations for MS-based analysis of the proteomic reactivity landscape.
... MS can also be used to map sites within protein interaction interfaces. Protein footprinting techniques, including most commonly used hydrogen deuterium exchange [46,47] and hydroxyl radical footprinting [48,49], can be used to probe in-solution differences in backbone interactions and side chain solvent accessibility, respectively, between different protein states. Within the gas-phase in the mass spectrometer, top-down fragmentation can additionally probe B factor or surface residues that reveal information on binding interactions and protein conformational states [50]. ...
Cyanobacteria, also known as blue–green algae, are ubiquitous organisms on the planet. They contain tremendous protein machineries that are of interest to the biotechnology industry and beyond. Recently, the number of annotated cyanobacterial genomes has expanded, enabling structural studies on known gene-coded proteins to accelerate. This review focuses on the advances in mass spectrometry (MS) that have enabled structural proteomics studies to be performed on the proteins and protein complexes within cyanobacteria. The review also showcases examples whereby MS has revealed critical mechanistic information behind how these remarkable machines within cyanobacteria function.
... Modification methods include dimethylation of lysine residues by formaldehyde and reducing reagent 24 , glycine ethyl ester (GEE)/EDC modification of carboxyl groups on aspartic and glutamic acid 25 , NHS-based modification of lysine residues using isobaric Tandem Mass Tag (TMT) labeling reagents 26 , isotopic succinic anhydride modification of lysine residues and isotopic N-ethylmaleimide (NEM) modification of cysteines 27 . Hydroxyl radical protein footprinting (HRPF), especially the fast photochemical oxidation of proteins (FPOP), coupled with MS has greatly advanced to permit the study of surface exposed regions of proteins in cell lysates and in vivo [28][29][30] . All of these MS-based methods involve reactions with solvent-accessible residues or surfaces, and are suitable for studying structural changes in proteins and complexes. ...
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry (qCLMS). Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å and use MS2 reporter ions for relative quantification of Qlinker-modified peptides. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints the key conformational and structural changes in biosensors and the RNA polymerase II (pol II) complex.
... An ideal radical dosimeter has a quantitative response to a broad effective radical dose and is easily measurable in real time to monitor HRPF reactions as they occur. Reviews of dosimetry methods using reporter peptides or small molecules are covered elsewhere [81,83]. For the high hydroxyl radical fluxes generated using FPOP, the first reported radical dosimeter with an optical readout was adenine [81]. ...
Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics. We describe here in this minireview the relatively new method of hydroxyl radical protein footprinting, a solution-state method that can provide structural and kinetic information on antibodies or antibody–antigen interactions useful for therapeutic antibody design. We provide a brief history of hydroxyl radical footprinting, examples of current implementations, and recent advances in throughput and accessibility.
