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.

Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.

MALDI Peptide Mapping for Fast Analysis in Protein Footprinting.

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting. The approach was applied to several model proteins that were submitted to two footprinting strategies, FPOP and GEE labeling, and their performance was evaluated. We found adequate coverage that can be improved with automatic off-line separation and spotting, demonstrating the capability to footprint accurately protein conformational change, showing that MALDI may be useful for selected applications in protein footprinting.

Pulsed Hydrogen-Deuterium Exchange Reveals Altered Structures and Mechanisms in the Aggregation of Familial Alzheimer's Disease Mutants.

Mutations of the Amyloid Precursor Protein, from which the amyloid ß peptide Aß42 is cleaved, are associated with familial Alzheimer's disease. The disease-relevant familial mutations include the Arctic (E22G), Iowa (D23N), Italian (E22K), Dutch (E22Q), Japanese (D7N), English (D6R), and Flemish (A21G) variants. A detailed mechanistic understanding of the aggregation behavior of the mutant peptides at the residue level is, however, still lacking. We report here a study of the aggregation kinetics of these mutants in vitro by pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a temporally and sequence resolved picture of their self-assembly. For all variants, HDX occurs to give a bimodal distribution representing two soluble classes of aggregates, one protected and one solvent-exposed. There is no evidence of other classes of structural intermediates within the detection limits of the HDX approach. The fractional changes in the bimodal exchange profiles for several regions of Aß42 reveal that the central and C-terminal peptides gain protection upon fibril formation, whereas the N-terminal regions remain largely solvent-accessible. For these mutants, all peptide fragments follow the same kinetics, acquiring solvent protection at the same time, further supporting that there are no significant populations of intermediate species under our experimental conditions. The results demonstrate the potential of pulsed HDX-MS for resolving the region-specific aggregation behavior of Aß42 isoforms in solution where X-ray crystallography and solid-state NMR (ssNMR) are challenged.

Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis.

The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts 'to mark' the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast photochemical oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a 'snapshot' of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography-tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

Composite Conformational Changes of Signaling Proteins upon Ligand Binding Revealed by a Single Approach: Calcium-Calmodulin Study.

Signaling proteins exemplified by calmodulin usually bind cooperatively to multiple ligands. Intermediate states and allosteric behavior are difficult to characterize. Here we extend a recently reported mass spectrometry (MS)-based method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) that characterizes complex binding systems typically found as signaling proteins. As reported previously, calmodulin's response to binding four Ca2+ can be determined by LITPOMS to reveal binding sites, binding order, and most importantly composite binding behavior. Modeling this behavior provides site-specific binding affinities. In this article, we dissect the composite, peptide-level conformational changes at several regions either by digestion with a different protease or by tandem MS of LITPOMS behavior at the amino-acid residue level. Such dissection greatly elevates spatial resolution and increases the confidence of binding-order assignment. These complementary views of complex protein conformational change recapitulate the cumulative understanding via a single approach, providing new insights on poorly understood yet important allostery and underpin an approach applicable for exploring other signaling systems.

Hydroxyl-Radical Reaction Pathways for the Fast Photochemical Oxidation of Proteins Platform As Revealed by 18O Isotopic Labeling.

Fast photochemical oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical (•OH) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of •OH oxidative modification of 13 amino acid residues by using 18O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O2), whereas the remaining residues take oxygen exclusively from O2. Results reported in this work deepen the understanding of •OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.

A Single Approach Reveals the Composite Conformational Changes, Order of Binding, and Affinities for Calcium Binding to Calmodulin.

We found that a newly developed method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) can characterize section-by-section of a protein the conformational changes induced by metal-ion binding. Peptide-level LITPOMS applied to Ca2+ binding to calmodulin reveals binding order and site-specific affinity, providing new insights on the behavior of proteins upon binding Ca2+. We established that EF hand-4 (EF-4) binds calcium first, followed by EF-3, EF-2, and EF-1 and determined the four affinity constants by modeling the extent-of-modification curves. We also found positive cooperativity between EF-4, EF-3 and EF-2, EF-1 and allostery involving the four EF-hands. LITPOMS recapitulates via one approach the calcium-calmodulin binding that required decades of sophisticated development to afford versatility, comprehensiveness, and outstanding spatial resolution.

A Fast Photochemical Oxidation of Proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins.

Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidation chemistry to prevent post-oxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed analysis. In addition to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO3-•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.

Protein-Ligand Interaction by Ligand Titration, Fast Photochemical Oxidation of Proteins and Mass Spectrometry: LITPOMS.

We report a novel method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin-calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution. Graphical Abstract.

Pulsed Hydrogen-Deuterium Exchange Illuminates the Aggregation Kinetics of α-Synuclein, the Causative Agent for Parkinson's Disease.

α-Synuclein (aS) forms toxic intermediates ranging from small oligomers and protofibrils to large amyloid fibrils. Understanding the time course of aS fibril formation and the role played by its regions is critical for therapeutic intervention. Here, we used pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) for the first time to probe kinetic intermediates of the full aS aggregation in vitro, achieving kinetic snapshots containing spatially resolved protein information about critical stages. Monitoring the resultant mass shifts shows distinct binomial abundances for two main exchange profiles: one that represents a fast-exchanging, solvent-accessible species and another with a more protected nature. We show using a series of proteolytic peptides from the full protein that self-association is most pronounced in the non-amyloid-ß-component region and less so for either terminus. The N-terminus, however, shows a minor protected population at mid- and late times, whereas the C-terminus shows predominantly unimodal HDX, indicating that these regions are devoid of any large conformational rearrangements. Focusing on the hydrophobic core, we confirmed and modeled the different isotopic distributions and calculated their relative fractions to discern their individual contributions. The data fitting reports respective t1/2 values, which are nearly identical and do not depend on location. We followed the aggregation by complementary transmission electron microscopy to observe the morphology of aggregates and circular dichroism to assess changes in secondary structure. Our results provide a detailed picture of aS aggregation in vitro and demonstrate that HDX-MS offers unique spatially resolved, coexisting kinetic intermediates in solution. This new platform is suitable for testing promising inhibitors of aS aggregation.

Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry.

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect ß-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen-Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3.

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 µM for the three different regions, giving an overall binding affinity of 7.4 µM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent Measurements.

Incorporation of a reporter peptide in solutions submitted to fast photochemical oxidation of proteins (FPOP) allows for the correction of adventitious scavengers and enables the normalization and comparison of time-dependent results. Reporters will also be useful in differential experiments to control for the inclusion of a radical-reactive species. This incorporation provides a simple and quick check of radical dosage and allows comparison of FPOP results from day-to-day and lab-to-lab. Use of a reporter peptide in the FPOP workflow requires no additional measurements or spectrometers while building a more quantitative FPOP platform. It requires only measurement of the extent of reporter-peptide modification in a LC/MS/MS run, which is performed by using either data-dependent scanning or an inclusion list. Graphical Abstract ᅟ.

Conformational-Sensitive Fast Photochemical Oxidation of Proteins and Mass Spectrometry Characterize Amyloid Beta 1-42 Aggregation.

Preventing and treating Alzheimer's disease require understanding the aggregation of amyloid beta 1-42 (Aß1-42) to give oligomers, protofibrils, and fibrils. Here we describe footprinting of Aß1-42 by hydroxyl radical-based fast photochemical oxidation of proteins (FPOP) and mass spectrometry (MS) to monitor the time-course of Aß1-42 aggregation. We resolved five distinct stages characterized by two sigmoidal behaviors, showing the time-dependent transitions of monomers-paranuclei-protofibrils-fibrillar aggregates. Kinetic modeling allows deciphering the amounts and interconversion of the dominant Aß1-42 species. Moreover, the irreversible footprinting probe provides insights into the kinetics of oligomerization and subsequent fibrillar growth by allowing the conformational changes of Aß1-42 at subregional and even amino-acid-residue levels to be revealed. The middle domain of Aß1-42 plays a major role in aggregation, whereas the N-terminus retains most of its solvent-accessibility during aggregation, and the hydrophobic C-terminus is involved to an intermediate extent. This approach affords an in situ, real-time monitoring of the solvent accessibility of Aß1-42 at various stages of oligomerization, and provides new insights on site-specific aggregation of Aß1-42 for a sample state beyond the capabilities of most other biophysical methods.

Protein Footprinting by Carbenes on a Fast Photochemical Oxidation of Proteins (FPOP) Platform.

Protein footprinting combined with mass spectrometry provides a method to study protein structures and interactions. To improve further current protein footprinting methods, we adapted the fast photochemical oxidation of proteins (FPOP) platform to utilize carbenes as the footprinting reagent. A Nd-YAG laser provides 355 nm laser for carbene generation in situ from photoleucine as the carbene precursor in a flow system with calmodulin as the test protein. Reversed-phase liquid chromatography coupled with mass spectrometry is appropriate to analyze the modifications produced in this footprinting. By comparing the modification extent of apo and holo calmodulin on the peptide level, we can resolve different structural domains of the protein. Carbene footprinting in a flow system is promising.

Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize CsgE oligomerization.

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE.

Dosimetry determines the initial OH radical concentration in fast photochemical oxidation of proteins (FPOP).

Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.