A Modular Variable Temperature FT-IMS Instrument Optimized for Gas-Phase Ion Chemistry Applications.

The latest iteration of modular, open-source rolled ion mobility spectrometers was characterized and tailored for heated ion chemistry experiments. Because the nature of ion-neutral interactions is innately linked to the temperature of the drift cell, heated IMS experiments explicitly probe the fundamental characteristics of these collisions. While classic mobility experiments examine ions through inert buffer gases, doping the drift cell with reactive vapor enables desolvated chemical reactions to be studied. By using materials with minimal outgassing and ensuring the isolation of the drift tube from the surrounding ambient conditions, an open-source drift cell outfitted with heating components enables investigations of chemical reactions as a function of temperature. We show here that elevated temperatures facilitate an increase in deuterium incorporation and allow for hydrogen/deuterium exchanges otherwise unattainable under ambient conditions. While the initial fast exchanges get faster as temperature is increased, the slow rate which rises from the kinetic nonlinearity though to be attributed to ion-neutral clustering, remains constant with no change in mobility shifts. Additionally, we show the analytical merit of multiplexing mobility data by comparing the performance of traditional signal-averaging and FT-IMS modes.

Distinct antithrombin activation modes for fondaparinux and natural heparins detected using millisecond hydrogen deuterium exchange and collision induced unfolding.

Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.

Probe capsid structure stability and dynamics of adeno-associated virus as an important viral vector for gene therapy by hydrogen-deuterium exchange-mass spectrometry.

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

Multidimensional LC-MS with 1D multi-method option and parallel middle-up and bottom-up MS acquisition for in-depth characterization of antibodies.

Monoclonal antibodies (mAbs) are large and highly heterogeneous species typically characterized using a plethora of analytical methodologies. There is a trend within the biopharmaceutical industry to combine several of these methods in one analytical platform to simultaneously assess multiple structural attributes. Here, a protein analyzer for the fully automated middle-up and bottom-up liquid chromatography-mass spectrometry (LC-MS) analysis of charge, size and hydrophobic variants is described. The multidimensional set-up combines a multi-method option in the first dimension (1D) (choice between size exclusion - SEC, cation exchange - CEX or hydrophobic interaction chromatography - HIC) with second dimension (2D) on-column reversed-phase (RPLC) based desalting, denaturation and reduction prior to middle-up LC-MS analysis of collected 1D peaks and parallel on-column trypsin digestion of denatured and reduced peaks in the third dimension (3D) followed by bottom-up LC-MS analysis in the fourth dimension (4D). The versatile and comprehensive workflow is applied to the characterization of charge, hydrophobic and size heterogeneities associated with an engineered Fc fragment and is complemented with hydrogen-deuterium exchange (HDX) MS and FcRn affinity chromatography - native MS to explain observations in a structural/functional context.

Protecting Lyophilized Escherichia coli Adenylate Kinase.

Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine Escherichia coli adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.

Structure of Human Serum Albumin at a Foam Surface.

Proteins can be adsorbed on the air-water interface (AWI), and the structural changes in proteins at the AWI are closely related to the foaming properties of foods and beverages. However, how these structural changes in proteins at the AWI occur is not well understood. We developed a method for the structural assessment of proteins in the foam state using hydrogen/deuterium exchange mass spectrometry. Adsorption sites and structural changes in human serum albumin (HSA) were identified in situ at the peptide-level resolution. The N-terminus and the loop (E492-T506), which contains hydrophobic amino acids, were identified as adsorption sites. Both the structural flexibility and hydrophobicity were considered to be critical factors for the adsorption of HSA at the AWI. Structural changes in HSA were observed after more than one minute of foaming and were spread widely throughout the structure. These structural changes at the foam AWI were reversible.

Synthesis of Deuterated Endochin-Like Quinolones.

Malaria continues to be a serious and debilitating disease. The emergence and spread of high-level resistance to multiple antimalarial drugs by Plasmodium falciparum has brought about an urgent need for new treatments that will be active against multidrug resistant malaria infections. One such treatment, ELQ-331 (MMV-167), an alkoxy carbonate prodrug of 4(1H)-quinolone ELQ-300, is currently in preclinical development with the Medicines for Malaria Venture. Clinical development of ELQ-331 or similar compounds will require the availability of isotopically labeled analogs. Unfortunately, a suitable method for the deuteration of these important compounds was not found in the literature. Here, we describe a facile and scalable method for the deuteration of 4(1H)-quinolone ELQ-300, its alkoxycarbonate prodrug ELQ-331, and their respective N-oxides using deuterated acetic acid.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Structural basis for the phase separation of the chromosome passenger complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Differentiation of isomeric chalcone and dihydroflavone using liquid chromatography coupled with hydrogen-deuterium exchange tandem mass spectrometry (HDX-MS/MS): An application for flavonoids-focused characterization of Snow chrysanthemum.

Although the co-occurrences of isomeric chalcones and dihydroflavones widely appear in medicinal plants, the differentiation of such isomerism seldom succeeds using MS/MS, attributing to totally identical MS/MS spectra. Here, efforts were paid to pursue an eligible tool allowing to address the technical challenge. Being inspired by that one more proton signal is observed in 1H NMR spectrum of isoliquiritigenin than liquiritigenin when employing DMSO­d6 as solvent, hydrogen-deuterium exchange (HDX)-MS/MS was evaluated towards differentiating isomeric chalcones and dihydroflavones through replacing H2O with D2O to prepare the mobile phase. As a result, differences were observed for either MS1 or MS2 spectrum when comparing two pairs of isomers, such as liquiritigenin vs. isoliquiritigenin and liquiritin vs. isoliquiritin, because the isomeric precursor and fragment ion species owned different amounts of hydroxyl protons and those reactive protons could be partially or completely substituted by deuterium protons at the exposure in D2O to result in n × 1.006 mass increments. Moreover, utmost four hydrogen/deuterium exchanges occurred for a single glucosyl moiety. Thereafter, HDX-MS/MS was applied to characterize the flavonoids of Snow chrysanthemum, a precious edible herbal medicine that is rich in isomeric chalcones and dihydroflavones. Through paying special attention to the deuterium labeling styles of (de)protonated molecules as well as those featured fragment ions, five pairs of isomeric chalcones and dihydroflavones were confirmatively differentiated, in addition to that 28 flavonoids were structurally annotated by applying those well-defined mass fragmentation rules. Hence, this study offered an in-depth insight towards the flavonoids-focused characterization of Snow chrysanthemum, and more importantly, HDX-MS/MS is a superior tool to differentiate, but not limited to, isomeric chalcones and dihydroflavones.

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

Probing Protein Complexes Composition, Stoichiometry, and Interactions by Peptide-Based Mass Spectrometry.

The characterization of a protein complex by mass spectrometry can be conducted at different levels. Initial steps regard the qualitative composition of the complex and subunit identification. After that, quantitative information such as stoichiometric ratios and copy numbers for each subunit in a complex or super-complex is acquired. Peptide-based LC-MS/MS offers a wide number of methods and protocols for the characterization of protein complexes. This chapter concentrates on the applications of peptide-based LC-MS/MS for the qualitative, quantitative, and structural characterization of protein complexes focusing on subunit identification, determination of stoichiometric ratio and number of subunits per complex as well as on cross-linking mass spectrometry and hydrogen/deuterium exchange as methods for the structural investigation of the biological assemblies.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4.

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463-645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange.

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

New insights into complex formation by SARS-CoV-2 nsp10 and nsp14.

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

Role of Hydroperoxyl Radicals in Heterogeneous Oxidation of Oxygenated Organic Aerosols.

Heterogeneous oxidative aging of organic aerosols (OA) occurs ubiquitously in the atmosphere, initiated by oxidants, such as the hydroxyl radicals (•OH). Hydroperoxyl radicals (HO2•) are also an important oxidant in the troposphere, and its gas-phase chemistry has been well studied. However, the role of HO2• in heterogeneous OA oxidation remains elusive. Here, we carry out •OH-initiated heterogeneous oxidation of several OA model systems under different HO2• conditions in a flow tube reactor and characterize the molecular oxidation products using a suite of mass spectrometry instrumentation. By using hydrogen-deuterium exchange (HDX) with thermal desorption iodide-adduct chemical ionization mass spectrometry, we provide direct observation of organic hydroperoxide (ROOH) formation from heterogeneous HO2• and peroxy radicals (RO2•) reactions for the first time. The ROOH may contribute substantially to the oxidation products, varied with the parent OA chemical structure. Furthermore, by regulating RO2• reaction pathways, HO2• also greatly influence the overall composition of the oxidized OA. Last, we suggest that the RO2• + HO2• reactions readily occur at the OA particle interface rather than in the particle bulk. These findings provide new mechanistic insights into the heterogeneous OA oxidation chemistry and help fill the critical knowledge gap in understanding atmospheric OA oxidative aging.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation.

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-ß component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.