Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition

Carter Lantz; Jaybree Lopez; Andrew K Goring; Muhammad A Zenaidee; Karl Biggs; Julian P Whitelegge; Rachel R Ogorzalek Loo; Frank-Gerrit Klärner; Thomas Schrader; Gal Bitan; Joseph A Loo

doi:10.1021/jasms.3c00281

. Author manuscript; available in PMC: 2024 Dec 6.

Published in final edited form as: J Am Soc Mass Spectrom. 2023 Nov 7;34(12):2739–2747. doi: 10.1021/jasms.3c00281

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition

Carter Lantz ¹, Jaybree Lopez ¹, Andrew K Goring ¹, Muhammad A Zenaidee ^1,², Karl Biggs ³, Julian P Whitelegge ⁴, Rachel R Ogorzalek Loo ¹, Frank-Gerrit Klärner ⁵, Thomas Schrader ⁵, Gal Bitan ^2,⁶, Joseph A Loo ^1,^6,^7,^*

¹Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, CA 90095 USA

²Australian Proteome Analysis Facility, Macquarie University, Macquarie Park, NSW, Australia

³Department of Neurology and Brain Research Institute, David Geffen School of Medicine at UCLA, University of California-Los Angeles, Los Angeles, CA 90095 USA

⁴The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California-Los Angeles, Los Angeles, CA 90095 USA

⁵Institute of Chemistry, University of Duisburg-Essen, Essen, Germany

⁶Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA, 90095 USA

⁷Department of Biological Chemistry, David Geffen School of Medicine at UCLA, University of California-Los Angeles, Los Angeles, CA 90095 USA

Authors

Carter Lantz − Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States

Jaybree Lopez− Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States

Andrew K. Goring − Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States

Muhammad A. Zenaidee - Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States; Australian Proteome Analysis Facility, Macquarie University, Macquarie Park, NSW, Australia

Karl Biggs − Department of Neurology and Brain Research Institute, David Geffen School of Medicine University of California, Los Angeles, Los Angeles, California 90095, United States

Julian Whitelegge − The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, United States

Frank-Gerrit Klärner - Institute of Chemistry, University of Duisburg-Essen, Essen, Germany

Thomas Schrader - Institute of Chemistry, University of Duisburg-Essen, Essen, Germany

Gal Bitan − Department of Neurology and Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, United States

Rachel R. Ogorzalek Loo − Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States

Corresponding Author: Joseph A. Loo − Department of Chemistry and Biochemistry and Department of Biological Chemistry, University of California Los Angeles, Los Angeles, California 90095, United States; JLoo@chem.ucla.edu

PMCID: PMC10959575 NIHMSID: NIHMS1977157 PMID: 37936057

Abstract

Parkinson’s disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small aromatic compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry of α-synuclein/CLR01 complexes pinpoint the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggests that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

Keywords: Native Mass Spectrometry, Top-Down Mass Spectrometry, Ion-Mobility Mass Spectrometry, Proteoform, α-Synuclein

Introduction

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are characterized by the formation of protein aggregates in the brain. These aggregates of proteins such as tau,¹ huntingtin,² and ataxin³ have been correlated with the death of brain neurons and the onset of neurodegenerative disease symptoms. α-Synuclein (α-syn) is a 14-kDa intrinsically disordered amyloid protein that has been proposed to regulate synapse formation in neurons.^{4, 5} In diseases called synucleinopathies, monomers of α-syn self-assemble into neurotoxic oligomers, which then transform into β-sheet-rich fibrils^{6, 7} and eventually form Lewy bodies and Lewy neurites in the brains of patients with Parkinson’s disease and dementia with Lewy bodies,⁸ or glial cytoplasmic inclusions in those of patients with multiple system atrophy. Although aggregation of α-syn has been studied extensively, the mechanistic reasons for this aggregation are not well understood, and a cure for these diseases has not been found.

Certain mutations and modifications have been found to modulate the rate of α-syn aggregation. For example, it has been reported that mutations leading to the amino-acid substitutions A30P and A53T increase the rate of α-syn oligomerization and aggregation.⁹ Post-translational modifications (PTMs), such as N-terminal acetylation have been reported to decrease the rate of α-syn aggregation^9–11 whereas phosphorylation at Ser-129 has been correlated with increased α-syn aggregation.¹² The binding of metal ions such as copper,¹³ manganese,¹⁴ and zinc¹⁵ have been linked to increased aggregation of α-syn. A shift in α-syn aggregation due to modifications is well documented. However, the reasons for the alteration in aggregation potential of the modified and metal-ion-bound α-syn are not well understood.

Despite extensive study, there is no therapy or cure for these synucleinopathies. A multitude of small molecule compounds have been found to inhibit fibril formation and dissociate existing fibrils.^16–20 Among these compounds is the molecular tweezer CLR01 that targets lysine and arginine residues and has been shown to inhibit α-syn self-assembly and toxicity in vitro and in vivo. Using mass spectrometry (MS), we have previously found that CLR01 binds various amyloid proteins^21–26 including α-syn. Molecular dynamic simulations suggested that CLR01 shifts the secondary structure of the N-17 domain of huntingtin²³ and compacts the tertiary structure of SOD1_bar²⁶ and α-syn,²¹ indicating that CLR01 shifts the conformational dynamics of amyloid protein. It was also found that 2 CLR01 binding events on α-syn induces an even greater structural shift.²¹ A shift in conformational dynamics due to CLR01 binding could favor conformations that are not aggregation prone so that monomers do not interact and form oligomers and fibrils. Building on previous research on CLR01 binding to α-syn, we asked here whether MS techniques could further illuminate the mechanism CLR01 utilizes to inhibit the self-assembly of the protein.

Over the past few decades, intact protein MS has become a useful technique to characterize proteoforms of amyloid proteins, including the determination of α-syn PTMs, such as oxidation,²⁷ acetylation,²⁸ and phosphorylation.^{29, 30} In addition, native MS has been shown to readily determine binding of noncovalent ligands, including CLR01^{22, 23} and metal ions,^{13, 31, 32} on amyloid proteins. MS analysis has been found to provide valuable insight into the types of modifications present on amyloid proteins and the extent of those modifications.

Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) MS can be used to obtain relevant information on proteins including amyloid proteins.³³ ECD is a nonergodic fragmentation technique that utilizes a beam of electrons to dissociate the peptide backbone without disrupting other intermolecular or intramolecular interactions.^34–36 ECD has been used for a variety of applications including protein sequencing, modification localization, and characterization of protein structure.³⁷ FT-ICR MS, a technique that can detect ions with superior mass accuracy and resolution,³⁸ is commonly utilized to provide high resolution ECD data for proteins³⁵ and protein complexes.³⁹ ECD in combination with FT-ICR MS has previously been found to pinpoint the binding of CLR01^21–25 and metal ions^{13, 32, 40} on various amyloid proteins including α-syn. Recent studies have shown that locating CLR01 binding with TD-MS can guide molecular dynamic simulations, which revealed that CLR01 binding can alter the structures of N17-Huntingin and α-syn proteins.^{21, 23}

Ion mobility MS (IM-MS) can be used to reveal relevant information on amyloid proteins and their complexes, such as size of the gas-phase ion.⁴¹ This technique has been used to record conformational differences of proteins due to factors such as solution conditions,⁴² collisional activation,^{43, 44} and protein charge.⁴⁵ Binding of ligands has also been reported to shift the IM drift time of proteins.^{46, 47} Previous IM-MS studies have also suggested that binding of various metal ions such as copper,^{13, 48} cobalt,³² manganese,³² calcium,⁴⁸ and zinc⁴⁸ alters the structure of α-syn. In other experiments, IM-MS revealed that small molecules such as CLR01,²² epigallocatechin-3-gallate (EGCG),¹⁶ and galic acid⁴⁹ induced structural changes in amyloid proteins. Moreover, IM-MS showed that detergent and lipid molecules such as DDM and PC-14 affected the structure of amyloid proteins.⁵⁰ PTMs such as oxidation⁵¹ and phosphorylation⁵² also affect the structure of amyloid proteins. IM-MS has also been utilized to study oligomer formation of amyloid proteins⁵³ and has been utilized to determine the modulation of oligomer formation using small molecules such as EGCG⁵⁴ and CLR01.⁵⁵

In this study, the interactions between molecular tweezer CLR01 and metal ions and various proteoforms of α-syn are characterized. Electron capture dissociation (ECD) reveals the location of all modifications, including the location of CLR01 binding to α-syn. In addition, IM-MS of α-syn/CLR01 complexes suggests that CLR01 compacts the structure of α-syn proteoforms, which may translate into a molecular mechanism that the molecular tweezer uses to reduce the aggregation of α-syn.

Experimental

For native MS experiments, 10–30 μM of α-syn or A30P α-syn (rPeptide, Watkinsville, GA) was dissolved into 20 mM ammonium acetate. Phosphorylated α-syn was obtained from Proteos Inc. (Kalamazoo, MI) and acetylated α-syn was synthesized as described previously.²⁸ CLR01, prepared as described previously,⁵⁶ was added at various amounts to obtain concentrations of 10–50 μM. For metal ion experiments, copper acetate was added at 10 μM and manganese acetate was added at 50 μM as these concentrations have previously been found to induce binding. UniDec was utilized to deconvolve the mass spectra.⁵⁷ Percentage of ligand binding was calculated by using the intensity of the unbound and bound peaks provided by the spectrum. Binding constants for three α-syn/CLR01 binding sites were obtained by fitting native MS data of α-syn titrated with CLR01 to a three-site Adair-Klotz model, which has been used to experimentally describe multiple non-identical binding sites of a ligand (L) to a single protein (P).⁵⁸ (See supplementary information)

Electrospray ionization-mass spectrometry (ESI-MS) experiments were performed using a 15T Solarix Fourier transform-ion cyclotron resonance (FT-ICR) instrument (Bruker Daltonics). The proteoforms were electrosprayed with an electrospray voltage of 1–2kV, a capillary exit voltage of 100V, a deflector plate voltage of 200V, a funnel voltage of 90V, and a skimmer voltage of 50V. Proteoforms of interest were isolated with notch values of 5–20 m/z. Protein ions were fragmented with ECD with a bias of 1V and a pulse length of 10ms to 50ms. The product ion spectra were deconvoluted with the SNAP algorithm from DataAnalysis (version 4.0 or 5.0) with a S/N limit of 2. c- and z-fragments were assigned with ClipsMS⁵⁹ using an error of 5ppm. CLR01, manganese, and copper were added as unlocalized modifications and the A30P α-syn substitution, phosphorylation at Ser-129, and N-terminal acetylation were added as localized modifications.

IM-MS data were acquired with a Synapt G2-Si quadrupole-IM-time-of-flight mass spectrometer (Waters). The spray voltage utilized was between 1–2kV, the sampling cone was set to 10–20V, and the source offset was set to 20V. IM specta were taken across the full m/z range and data for each charge state was extracted over the [M+nH]ⁿ⁺ peak. IM-MS of CLR01 binding to wild-type (WT) and A30P α-syn was acquired with CLR01 added to the solution at a 5x molar concentration. IM-MS of acetylated and phosphorylated α-syn bound with CLR01 were analyzed at a 1:1 molar ratio. IM-MS of manganese and copper bound α-syn bound with CLR01 were analyzed at a 1:3 molar ratio.

Results and Discussion

CLR01 Binding to α-Syn Proteoforms

Previous research found that CLR01 binds to wild type (WT) α-syn when combined at a 1:1 molar ratio;²¹ however, it is known that greater inhibition of fibril formation can be achieved at higher concentrations of CLR01.¹⁸ To investigate the interaction between CLR01 and α-syn at higher concentrations of CLR01, CLR01 was added to a native solution of WT α-syn at a 5x molar concentration. The resulting mass spectrum revealed a broad charge state distribution characteristic of intrinsically disordered proteins and multiple CLR01 binding events (Fig. 1). The deconvolved mass spectrum indicated that up to 5 binding events can be observed and 63% of all CLR01 in solution is bound to the protein. The 5^th binding site was of quite low abundance, however the peaks as well as their sodiated proteoforms could be resolved (Fig. S1). A solution of A30P α-syn, a proteoform that has been correlated with increased aggregation,⁹ and CLR01 were also electrosprayed in native solution conditions, revealing that multiple CLR01 molecules can bind to A30P α-syn (Fig. S2), and up to 5 binding events can be observed on α-syn representing 74% of total CLR01. The increased number of CLR01 binding events at higher concentrations of CLR01 could be a reason that α-syn aggregation is further inhibited at those increased concentrations.

Figure 1. — A native mass spectrum of α-synuclein with a 5x molar ratio of CLR01. The deconvolved mass spectrum (inset) reveals up to 5 CLR01 binding events can be observed.

Mass spectra were also collected at lower molar ratios of WT α-syn:CLR01. These spectra were utilized to calculate K_d values for each binding event (Fig. S3). Intensity values for all α-syn/CLR01 complexes are located in Table S1. It was found that the first binding event had a K_d of 47 μM. The K_d values for the second and third binding sites were 70 μM and 85 μM, respectively. These three sites are presumed to bind to specific regions of the protein sequence. The K_d values for the fourth and fifth binding sites did not converge well and are thought to bind nonspecifically to other regions of the protein.

It is known that some PTMs can modulate the rate of amyloid protein aggregation, including N-terminal acetylation and Ser-129 phosphorylation. An equimolar mixture of wild type α-syn, acetylated α-syn, and phosphorylated α-syn was measured by native MS (Fig. S4A). Each proteoform could be resolved in the native mass spectrum, allowing for accurate quantification of each proteoform. Next, an equimolar ratio of CLR01 was added to the α-syn solution (Fig. S4B). The spectrum revealed multiple CLR01 molecules bound to all proteoforms present in the sample. The deconvolved mass spectrum shows up to 2 CLR01 molecules can bind each proteoform under these conditions. Comparing peak intensity ratios of unbound and CLR01 bound states of α-syn indicated that CLR01 binds each α-syn proteoform with a similar affinity. Table S2 contains the intensity values for each proteoform present in the spectrum.

CLR01 Binding to α-Syn/Metal Complexes

Mn²⁺ binding to α-syn has been shown to increase the rate of α-syn aggregation.¹⁴ As α-syn could be metal-bound in the brain, we sought to determine the relationship between α-syn and the binding of Mn²⁺ and CLR01. nMS was performed on a 1:5:3 molar ratio solution of α-syn to Mn²⁺ to CLR01, respectively. The resulting spectrum indicated multiple binding events of both Mn²⁺ and CLR01 (Fig. 2A), with up to 5 Mn²⁺ and 4 CLR01 molecule binding events observed. In addition, the mass spectrum suggests that binding of CLR01 enhances Mn²⁺ binding. When CLR01 is not bound to the protein, 86% of the α-syn was bound by Mn²⁺. However, when 1 CLR01 molecule was bound, 89% of the α-syn was bound by Mn²⁺, and when 2 CLR01 molecules were bound, 92% of the α-syn was bound by Mn²⁺. There was some α-syn that contained 3 CLR01 molecules, but the signal-to- noise was quite low and the peaks could not be accurately quantified. Table S3 contains intensity values for all the proteoforms present in the spectrum.

Figure 2. — Native mass spectra of A) α-synuclein with a 5x molar ratio of manganese and a 3x molar ratio of CLR01 and B) α-synuclein with a 1x molar ratio of copper and a 3x molar ratio of CLR01. The deconvolved mass spectra (insets) reveal that multiple manganese ions bind α-synuclein while only 1 copper ion binds α-synuclein. In addition, multiple CLR01 molecules bind to the α-synuclein/metal ion complexes.

Likewise, Cu²⁺ binding also increases the rate of α-syn aggregation.¹³ nMS of a α-syn/ Cu²⁺/CLR01 solution at a 1:1:3 molar ratio shows multiple CLR01 binding sites but only 1 copper binding event (Fig. 2B). This observation contrasts with the multiple-binding data for Mn²⁺ (Fig. 2A). Up to 3 CLR01 molecules can bind α-syn simultaneously with Cu²⁺ ions bound. Comparison of intensities reveals that when 0 CLR01 molecules are bound to the protein, copper binds 65% of the α-syn; however when 3 CLR01 molecules bind, 57% of α-syn is bound by Cu²⁺. Multiple CLR01 binding events compete with Cu²⁺ binding on the protein. Peaks corresponding to binding of 4 CLR01 molecules were present in the spectrum, although the signal-to-noise was quite low and could not be accurately quantified. Table S4 contains the intensity values for all the proteoforms present in the spectrum. The reasons for the opposite trends observed for Mn²⁺ and Cu²⁺ are unclear, but the data suggests that CLR01 binding modulates metal binding through structural effects at the protein level.

Top-Down MS Analysis of CLR01-/Metal-Binding to α-Syn

To further characterize these protein/CLR01 complexes, we performed ECD of numerous complexes. ECD was performed due to the technique’s ability to preserve the interaction between noncovalent ligand and the protein. Collisionally activated dissociation (CAD) has been performed on α-syn with noncovalent ligands attached,³² but the ligand tends to dissociate from the protein during the fragmentation process. Our lab has previously performed ECD of the WT α-syn/CLR01 complex to determine that CLR01 binds predominantly to K10 and/or K12.²¹ However, it is not known where other CLR01 molecules bind when multiple CLR01 molecules are bound to α-syn. The molecular tweezer targets available Lys and Arg resiudes, and therefore other basic sites on α-syn should be recognized with higher molar ratios of CLR01. To determine the location of the second CLR01 binding site, the 13+ charge state of wildtype α-syn bound with 2 CLR01 molecules (m/z 1223.85) was isolated and fragmented by ECD (Fig. 3A). The ECD spectrum revealed product ions that are not bound to CLR01, and product ions with 1 and 2 CLR01 molecules bound. Product ion assignments indicate that the second CLR01 molecule binds to K12 with a secondary binding location at K21. ECD performed on the A30P α-syn/CLR01 complex showed CLR01 binding at K10 or K12 (Fig. 3B), similar to WT α-syn. ECD of A30P α-syn with 2 CLR01 molecules bound indicates that CLR01 binds K10 and K12 with a possible additional site of binding at K21 (Fig. S5A). Thus, the A30P mutation does not significantly alter the structure of α-syn to change the binding of CLR01. To further study the impact of post-translational modifications on the binding site(s) of CLR01 on α-syn, ECD was performed on the acetylated and phosphorylated proteoforms of α-syn in the presence of CLR01. ECD of the acetylated α-syn/CLR01 complex showed product ions that contained the N-terminal acetylation site and the CLR01 molecule (Fig. S5B), with K21 as the primary binding site of CLR01. It is possible the binding site of CLR01 may shift slightly due to the stabilization of the α-helical structure of the N-terminus by acetylation. In contrast, ECD fragmentation of the phosphorylated α-syn at S129 bound to CLR01 confirmed phosphorylation at S129 and the location of CLR01 binding at K10 or K12, which is similar to wild type α-syn (Fig. S5C). These results indicate that CLR01 still binds (mostly) to the N-terminus of α-syn for all proteoforms studied.

Figure 3. — ECD fragmentation spectra for 13+ charge state of A) the α-syn/CLR01/CLR01 complex, B) the A30P α-syn/CLR01 complex, and C) the α-syn/Mn²⁺/CLR01 complex with the corresponding fragment location maps that reveal the location of the modifications and the CLR01 binding location (on the N-terminus of the protein). The vertical black dotted lines represent K10, K12, and K21, which are possible binding sites of CLR01.

Previous studies have utilized TD-MS to determine the binding site(s) of metal ions such as copper¹³ and manganese³² on α-syn. To further characterize the interaction between α-syn, metal ions, and CLR01 molecules, ECD fragmentation was performed on the α-syn/Mn²⁺/CLR01 complex. CLR01 binds to K10 or K12 of the protein and Mn²⁺ binds to the acidic C-terminal region of α-syn (Fig. 3C). This is consistent with previous α-syn binding studies with each ligand monitored separately. ECD of the α-syn/Cu²⁺/CLR01 complex revealed peaks bound with Cu²⁺, CLR01, and both Cu²⁺ and CLR01. Analysis of the spectrum reveals that CLR01 binds to K10 and Cu²⁺ binds to the N-terminus of the protein (Fig. S5D), which again is consistent with previous work studying the ligands separately. The fact that both of these ligands bind at or near the N-terminus may explain why Cu²⁺ and CLR01 compete for binding when multiple CLR01 molecules are bound. TD-MS with ECD of protein/ligand/metal complexes can accurately identify the location of the ligands and may explain why binding of metal ions is modulated by CLR01 binding.

ECD data of CLR01 bound α-syn reveals CLR01 binds to the N-terminus of the protein. Our data indicates that the most likely residues for binding are K10, K12, and K21; however, the fragmentation reveals that CLR01 could bind to many lysine residues on the basic N-terminus as is evident by the presence of numerous unbound fragments containing the N-terminus. An NMR structure of micelle bound α-syn reveals two α-helixes that provide a loose structure for the N-terminus.⁶⁰ It is possible that the N-terminus forms a loose structure providing a pocket containing many lysines that CLR01 can bind. CLR01 binding in this region may shift the conformational dynamics of the protein and prevent aggregation of α-syn monomers.

Ion-Mobility Analysis of CLR01 Binding to α-Syn/Metal Complexes

Ion-mobility mass spectrometry (IM-MS) has revealed that small molecule-binding can alter the structure of amyloid proteins. Previous research has found that CLR01 compacts the structure of tau protein upon binding.²² Similarly, it has also been found that the natural product, EGCG, promotes compaction of α-syn structure upon binding.¹⁶ Thus, IM-MS was performed to determine the potential structural changes of α-syn upon CLR01 binding. IM-MS of the 9+ charge state of WT α-syn revealed the existence of 2 abundant ensembles of ions (Fig. 4A). The mobiligram distribution of the 9+ charge state is quite similar to previously published data.^{13, 32}

Figure 4. — The ion mobiligrams for the 9+ charge state of A) unbound and B) CLR01-bound WT α-syn; C) unbound and D) CLR01-bound phosphorylated α-syn; and E) unbound and F) CLR01-bound α-syn/ Cu²⁺. CLR01 binding results in an increased abundance of early arriving ensembles of α-syn.

IM-MS of the 9+ charge state with 1 CLR01 molecule bound reveals an increase in abundance of the early arriving ensemble and the presence of two additional early arriving ensembles (Fig. 4B). Additional CLR01 binding further promotes an increase in the abundance of early arriving ensembles (Fig. S6A). The 8+ charge state also shows an increase of early arriving species (Fig. S6B), whereas the higher charge states such as 12+ do not show significant shifts in the mobiligram when CLR01 binds (Fig. S6C).

IM-MS of the A30P α-syn/CLR01 complexes showed a similar pattern. The 8+ and 9+ charge states of A30P α-syn revealed multiple ensembles, and when CLR01 binds, the early arriving ensembles increase in abundance. (Figs. S7A,B) The mobiligram of the 12+ charge state of A30P α-syn does not change significantly upon CLR01 binding (Fig. S7C). The increase in abundance of early arriving ensembles for the 8+ and 9+ charge states due to CLR01 binding suggests that CLR01 compacts the structure of α-syn. It is possible this compaction is due to the presence of salt bridges formed by the negatively charged phosphates on CLR01 and positively charged residues of α-syn. The features for the higher charge states do not shift considerably, possibly due to an accumulation of charge that extends the structure of the protein owing to charge repulsion between adjacent charge sites.^{61, 62}

To further investigate the structural effects of CLR01 binding, IM-MS was performed on the CLR01 unbound and bound forms of phosphorylated and acetylated α-syn. The 9+ IM profile of phosphorylated α-syn also revealed 4 ensembles of the protein (Fig. 4C). Interestingly, the ion mobiligram for phosphorylated α-syn reveals increasing early (compact) arriving conformers relative to WT α-syn. Compaction upon phosphorylation has been reported for peptides⁶³ and proteins⁵² previously. IM-MS of CLR01-bound phosphorylated α-syn showed further increase in abundance of the early arriving ensembles (Fig. 4D). The 8+ charge state (Fig. S8A) showed similar results to the 9+ charge state; however, the higher charge states such as 12+ did not shift significantly when bound with CLR01 (Fig. S8B).

The 9+ IM profile of acetylated α-syn also revealed 4 ensembles of ions and the CLR01 bound state showed an increase in the abundance of early arriving ensembles indicating CLR01 compacts the structure of acetylated α-syn (Fig. S9A). This observation is also observed for the 8+ charge state (Fig. S9B), whereas the 12+ charge state did not shift significantly (Fig. S9C). These data suggests that CLR01 compacts the structure of phosphorylated and acetylated proteoforms much like WT α-syn.

Previous IM-MS research from our lab found that Cu²⁺ binding increases the early arriving ensembles of α-syn.¹³ IM-MS was performed on the unbound and CLR01 bound forms of the α-syn/ Cu²⁺ complex. The 9+ charge state of Cu²⁺ bound α-syn revealed 4 ensembles of ions with early arriving ensembles significantly more abundant than observed for the WT form (Fig. 4E). CLR01-binding to the α-syn/ Cu²⁺ complex further increases the abundance of the early arriving conformers (Fig. 4F); the 8+ charge state showed similar trends (Fig. S10A). The IM-MS profiles for the 12+ charge state as expected did not shift significantly when bound to either Cu²⁺ or CLR01 (Fig. S10B). It is possible that compaction of the α-syn structure due to CLR01 binding could shield the region of Cu²⁺ binding, which is consistent with the observation that Cu²⁺ binding appears to be inhibited when multiple CLR01 molecules are bound. Mn²⁺ binding only slightly compacts α-syn; however, when CLR01 and Mn²⁺ both bind, the structure of α-syn is compacted (Fig. S11).

Compaction of α-syn due to CLR01 binding could be a possible mechanism that the molecular tweezer utilizes for inhibiting oligomer and fibril formation. The disordered nature of the N-terminus of WT α-syn could yield extended structures that expose the aggregation region and provide the opportunity for monomers to interact. Our data suggests that multiple CLR01 molecules bind to the N-terminus of α-syn proteoforms and compacts its structure. This compaction of the N-terminus could shield the aggregation region of α-syn from other monomers, resulting in a decreased probability for proteins to aggregate and form oligomers and fibrils.

Conclusion

In this study we characterized how the molecular tweezer, CLR01, interacts with various α-syn proteoforms, including its metal-bound forms. CLR01-binding modulates the binding of Cu²⁺ and Mn²⁺. Top-down MS fragmentation with ECD provided information on the location of the modifications for all proteoforms and the binding location of CLR01 along the protein sequence. For all α-syn forms studied, CLR01 binds to the N-terminal region of the α-syn polypeptide. IM-MS of various α-syn/CLR01 complexes indicates that CLR01 binding compacts the structure of the protein. This observation closely resembles the effect EGCG binding has on α-syn.¹⁶ Characterization of CLR01 and EGCG binding to α-syn could indicate a mechanism that small molecules can utilize to inhibit fibril formation. Compaction could shield the region prone to aggregation from binding to other monomers. Preventing the formation of oligomers and fibrils could halt the death of brain neurons and the onset of neurodegenerative diseases.

Supplementary Material

JASMS SI 2023

NIHMS1977157-supplement-JASMS_SI_2023.pdf^{(1.7MB, pdf)}

ACKNOWLEDGMENT

J.A.L. and R.R.O.L. acknowledge support from the US National Institutes of Health (R01GM103479, R35GM145286, S10RR028893, S10OD018504) and the US Department of Energy (DE-FC02-02ER63421). C.L. acknowledges support from the Ruth L. Kirschstein National Research Service Award program (GM007185). A.G. acknowledges support from the National Institute of Dental & Craniofacial Research (T90DE030860). G.B. acknowledges support from the National Institute of Neurological Disorders and Stroke (RF1NS126406 and R21NS130326), the Cure Sanfilippo Foundation (20215318), and the Binder Foundation.

Footnotes

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Additional details on determining K_D values; native mass spectra of A30P α-syn, acetylated α-syn, and phosphorylated α-syn binding to CLR01; fragment location maps from native top-down MS of A30P α-syn, acetylated α-syn, and phosphorylated α-syn binding to CLR01; ion mobility profiles of wild-type α-syn, A30P α-syn, acetylated α-syn, and phosphorylated α-syn bound to CLR01; ion mobility profiles of α-syn bound to copper and manganese; tables listing the molecular masses and relative abundances of wild-type α-syn, A30P α-syn, acetylated α-syn, and phosphorylated α-syn bound to CLR01, copper, and manganese.

References

1.Johnson VE; Stewart W; Smith DH, Widespread tau and amyloid-beta pathology many years after a single traumatic brain injury in humans. Brain pathology 2012, 22 (2), 142–149. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Díaz-Hernández M; Moreno-Herrero F; Gómez-Ramos P; Morán MA; Ferrer I; Baró AM; Avila J; Hernández F; Lucas JJ, Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain. Journal of Neuroscience 2004, 24 (42), 9361–9371. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Yamada M; Hayashi S; Tsuji S; Takahashi H, Involvement of the cerebral cortex and autonomic ganglia in Machado-Joseph disease. Acta neuropathologica 2001, 101 (2), 140–144. [DOI] [PubMed] [Google Scholar]
4.Lautenschläger J; Stephens AD; Fusco G; Ströhl F; Curry N; Zacharopoulou M; Michel CH; Laine R; Nespovitaya N; Fantham M, C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction. Nature communications 2018, 9 (1), 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Bellani S; Sousa VL; Ronzitti G; Valtorta F; Meldolesi J; Chieregatti E, The regulation of synaptic function by α-synuclein. Communicative & Integrative Biology 2010, 3 (2), 106–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Schweighauser M; Shi Y; Tarutani A; Kametani F; Murzin AG; Ghetti B; Matsubara T; Tomita T; Ando T; Hasegawa K, Structures of α-synuclein filaments from multiple system atrophy. Nature 2020, 1–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Li B; Ge P; Murray KA; Sheth P; Zhang M; Nair G; Sawaya MR; Shin WS; Boyer DR; Ye S, Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel. Nature communications 2018, 9 (1), 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Baba M; Nakajo S; Tu P-H; Tomita T; Nakaya K; Lee V; Trojanowski JQ; Iwatsubo T, Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. The American journal of pathology 1998, 152 (4), 879. [PMC free article] [PubMed] [Google Scholar]
9.Stephens AD; Zacharopoulou M; Moons R; Fusco G; Seetaloo N; Chiki A; Woodhams PJ; Mela I; Lashuel HA; Phillips JJ, Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity. Nature Communications 2020, 11 (1), 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Kang L; Moriarty GM; Woods LA; Ashcroft AE; Radford SE; Baum J, N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Science 2012, 21 (7), 911–917. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Bu B; Tong X; Li D; Hu Y; He W; Zhao C; Hu R; Li X; Shao Y; Liu C, N-terminal acetylation preserves α-synuclein from oligomerization by blocking intermolecular hydrogen bonds. ACS Chemical Neuroscience 2017, 8 (10), 2145–2151. [DOI] [PubMed] [Google Scholar]
12.Sato H; Arawaka S; Hara S; Fukushima S; Koga K; Koyama S; Kato T, Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson’s disease. Journal of Neuroscience 2011, 31 (46), 16884–16894. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Choi TS; Lee J; Han JY; Jung BC; Wongkongkathep P; Loo JA; Lee MJ; Kim HI, Supramolecular modulation of structural polymorphism in pathogenic α-synuclein fibrils using copper (II) coordination. Angewandte Chemie International Edition 2018, 57 (12), 3099–3103. [DOI] [PubMed] [Google Scholar]
14.Uversky VN; Li J; Fink AL, Metal-triggered structural transformations, aggregation, and fibrillation of human α-synuclein a possible molecular link between parkinson′ s disease and heavy metal exposure. Journal of Biological Chemistry 2001, 276 (47), 44284–44296. [DOI] [PubMed] [Google Scholar]
15.Byrd EJ; Wilkinson M; Radford SE; Sobott F, Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein. Journal of the American Society for Mass Spectrometry 2023, 34 (3), 493–504. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Konijnenberg A; Ranica S; Narkiewicz J; Legname G; Grandori R; Sobott F; Natalello A, Opposite structural effects of epigallocatechin-3-gallate and dopamine binding to α-synuclein. Analytical chemistry 2016, 88 (17), 8468–8475. [DOI] [PubMed] [Google Scholar]
17.Hong D-P; Fink AL; Uversky VN, Structural characteristics of α-synuclein oligomers stabilized by the flavonoid baicalein. Journal of molecular biology 2008, 383 (1), 214–223. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Prabhudesai S; Sinha S; Attar A; Kotagiri A; Fitzmaurice AG; Lakshmanan R; Ivanova MI; Loo JA; Klärner F-G; Schrader T, A novel “molecular tweezer” inhibitor of α-synuclein neurotoxicity in vitro and in vivo. Neurotherapeutics 2012, 9 (2), 464–476. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Bieschke J; Russ J; Friedrich RP; Ehrnhoefer DE; Wobst H; Neugebauer K; Wanker EE, EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proceedings of the National Academy of Sciences 2010, 107 (17), 7710–7715. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Xu M; Loa-Kum-Cheung W; Zhang H; Quinn RJ; Mellick GD, Identification of a new α-synuclein aggregation inhibitor via mass spectrometry based screening. ACS chemical neuroscience 2019, 10 (6), 2683–2691. [DOI] [PubMed] [Google Scholar]
21.Acharya S; Safaie BM; Wongkongkathep P; Ivanova MI; Attar A; Klärner F-G; Schrader T; Loo JA; Bitan G; Lapidus LJ, Molecular basis for preventing α-synuclein aggregation by a molecular tweezer. Journal of Biological Chemistry 2014, 289 (15), 10727–10737. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Nshanian M; Lantz C; Wongkongkathep P; Schrader T; Klärner F-G; Blümke A; Despres C; Ehrmann M; Smet-Nocca C; Bitan G, Native top-down mass spectrometry and ion mobility spectrometry of the interaction of tau protein with a molecular tweezer assembly modulator. Journal of the American Society for Mass Spectrometry 2018, 30 (1), 16–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Vöpel T; Bravo-Rodriguez K; Mittal S; Vachharajani S; Gnutt D; Sharma A; Steinhof A; Fatoba O; Ellrichmann G; Nshanian M, Inhibition of huntingtin exon-1 aggregation by the molecular tweezer CLR01. Journal of the American Chemical Society 2017, 139 (16), 5640–5643. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Malik R; Meng H; Wongkongkathep P; Corrales CI; Sepanj N; Atlasi RS; Klärner F-G; Schrader T; Spencer MJ; Loo JA, The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS. Journal of Biological Chemistry 2019, 294 (10), 3501–3513. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Sinha S; Lopes DH; Du Z; Pang ES; Shanmugam A; Lomakin A; Talbiersky P; Tennstaedt A; McDaniel K; Bakshi R, Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. Journal of the American Chemical Society 2011, 133 (42), 16958–16969. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Samanta N; Ruiz-Blanco YB; Fetahaj Z; Gnutt D; Lantz C; Loo JA; Sanchez-Garcia E; Ebbinghaus S, Superoxide dismutase 1 folding stability as a target for molecular tweezers in SOD1-related amyotrophic lateral sclerosis. ChemBioChem 2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Leong SL; Pham CL; Galatis D; Fodero-Tavoletti MT; Perez K; Hill AF; Masters CL; Ali FE; Barnham KJ; Cappai R, Formation of dopamine-mediated α-synuclein-soluble oligomers requires methionine oxidation. Free Radical Biology and Medicine 2009, 46 (10), 1328–1337. [DOI] [PubMed] [Google Scholar]
28.Sarafian TA; Ryan CM; Souda P; Masliah E; Kar UK; Vinters HV; Mathern GW; Faull KF; Whitelegge JP; Watson JB, Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein. PLoS One 2013, 8 (5), e63557. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Kellie JF; Higgs RE; Ryder JW; Major A; Beach TG; Adler CH; Merchant K; Knierman MD, Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson’s disease brain tissue by intact protein mass spectrometry. Scientific reports 2014, 4, 5797. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Dutta S; Hornung S; Taha HB; Biggs K; Siddique I; Chamoun LM; Shahpasand-Kroner H; Lantz C; Herrera-Vaquero M; Stefanova N, Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples. ACS Chemical Neuroscience 2023, 14 (7), 1238–1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Ly T; Julian RR, Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry. Journal of the American Society for Mass Spectrometry 2011, 19 (11), 1663–1672. [DOI] [PubMed] [Google Scholar]
32.Wongkongkathep P; Han JY; Choi TS; Yin S; Kim HI; Loo JA, Native top-down mass spectrometry and ion mobility MS for characterizing the cobalt and manganese metal binding of α-synuclein protein. Journal of the American Society for Mass Spectrometry 2018, 29 (9), 1870–1880. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Taha HB; Chawla E; Bitan G, IM-MS and ECD-MS/MS Provide Insight into Modulation of Amyloid Proteins Self-Assembly by Peptides and Small Molecules. Journal of the American Society for Mass Spectrometry 2023. [DOI] [PubMed] [Google Scholar]
34.Haselmann KF; Jørgensen TJ; Budnik BA; Jensen F; Zubarev RA, Electron capture dissociation of weakly bound polypeptide polycationic complexes. Rapid communications in mass spectrometry 2002, 16 (24), 2260–2265. [DOI] [PubMed] [Google Scholar]
35.Shi SD-H; Hemling ME; Carr SA; Horn DM; Lindh I; McLafferty FW, Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry. Analytical Chemistry 2001, 73 (1), 19–22. [DOI] [PubMed] [Google Scholar]
36.Zhurov KO; Fornelli L; Wodrich MD; Laskay ÜA; Tsybin YO, Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chemical Society Reviews 2013, 42 (12), 5014–5030. [DOI] [PubMed] [Google Scholar]
37.Zhou M; Lantz C; Brown KA; Ge Y; Paša-Tolić L; Loo JA; Lermyte F, Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry. Chemical science 2020, 11 (48), 12918–12936. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Comisarow MB; Marshall AG, Resolution-enhanced Fourier transform ion cyclotron resonance spectroscopy. The Journal of Chemical Physics 1975, 62 (1), 293–295. [Google Scholar]
39.Li H; Wongkongkathep P; Van Orden SL; Ogorzalek Loo RR; Loo JA, Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment. Journal of the American Society for Mass Spectrometry 2014, 25 (12), 2060–2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Lermyte F; Everett J; Lam YP; Wootton CA; Brooks J; Barrow MP; Telling ND; Sadler PJ; O’Connor PB; Collingwood JF, Metal ion binding to the amyloid β monomer studied by native top-down FTICR mass spectrometry. Journal of the American Society for Mass Spectrometry 2019, 30 (10), 2123–2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Lanucara F; Holman SW; Gray CJ; Eyers CE, The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nature chemistry 2014, 6 (4), 281–294. [DOI] [PubMed] [Google Scholar]
42.Shi H; Atlasevich N; Merenbloom SI; Clemmer DE, Solution dependence of the collisional activation of ubiquitin [M+ 7H] 7+ ions. Journal of the American Society for Mass Spectrometry 2014, 25 (12), 2000–2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Dixit SM; Polasky DA; Ruotolo BT, Collision induced unfolding of isolated proteins in the gas phase: past, present, and future. Current opinion in chemical biology 2018, 42, 93–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Bernstein SL; Liu D; Wyttenbach T; Bowers MT; Lee JC; Gray HB; Winkler JR, α-Synuclein: Stable compact and extended monomeric structures and pH dependence of dimer formation. Journal of the American Society for Mass Spectrometry 2004, 15 (10), 1435–1443. [DOI] [PubMed] [Google Scholar]
45.Jhingree JR; Beveridge R; Dickinson ER; Williams JP; Brown JM; Bellina B; Barran PE, Electron transfer with no dissociation ion mobility–mass spectrometry (ETnoD IM-MS). The effect of charge reduction on protein conformation. International Journal of Mass Spectrometry 2017, 413, 43–51. [Google Scholar]
46.Williams JP; Brown JM; Campuzano I; Sadler PJ, Identifying drug metallation sites on peptides using electron transfer dissociation (ETD), collision induced dissociation (CID) and ion mobility-mass spectrometry (IM-MS). Chemical communications 2010, 46 (30), 5458–5460. [DOI] [PubMed] [Google Scholar]
47.Campuzano ID; Lippens JL, Ion mobility in the pharmaceutical industry: an established biophysical technique or still niche? Current Opinion in Chemical Biology 2018, 42, 147–159. [DOI] [PubMed] [Google Scholar]
48.Moons R; Konijnenberg A; Mensch C; Van Elzen R; Johannessen C; Maudsley S; Lambeir A-M; Sobott F, Metal ions shape α-synuclein. Scientific reports 2020, 10 (1), 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Liu Y; Carver JA; Calabrese AN; Pukala TL, Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 2014, 1844 (9), 1481–1485. [DOI] [PubMed] [Google Scholar]
50.Moons R; van der Wekken-de Bruijne R; Maudsley S; Lemière F; Lambeir A-M; Sobott F, Effects of Detergent on α-Synuclein Structure. A Native MS-Ion Mobility Study. International Journal of Molecular Sciences 2020, 21 (21), 7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Ponzini E; De Palma A; Cerboni L; Natalello A; Rossi R; Moons R; Konijnenberg A; Narkiewicz J; Legname G; Sobott F, Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure. Journal of Biological Chemistry 2019, 294 (14), 5657–5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Lantz C In Structural Analysis of Gas-Phase Phosphoproteins, American Society for Mass Spectrometry, Atlanta, GA, Atlanta, GA, 2019. [Google Scholar]
53.Bernstein SL; Dupuis NF; Lazo ND; Wyttenbach T; Condron MM; Bitan G; Teplow DB; Shea J-E; Ruotolo BT; Robinson CV, Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nature chemistry 2009, 1 (4), 326–331. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Bleiholder C; Do TD; Wu C; Economou NJ; Bernstein SS; Buratto SK; Shea J-E; Bowers MT, Ion mobility spectrometry reveals the mechanism of amyloid formation of Aβ (25–35) and its modulation by inhibitors at the molecular level: epigallocatechin gallate and scyllo-inositol. Journal of the American Chemical Society 2013, 135 (45), 16926–16937. [DOI] [PubMed] [Google Scholar]
55.Zheng X; Liu D; Klärner F-G; Schrader T; Bitan G; Bowers MT, Amyloid β-protein assembly: the effect of molecular tweezers CLR01 and CLR03. The Journal of Physical Chemistry B 2015, 119 (14), 4831–4841. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Talbiersky P; Bastkowski F; Klärner F-G; Schrader T, Molecular clip and tweezer introduce new mechanisms of enzyme inhibition. Journal of the American Chemical Society 2008, 130 (30), 9824–9828. [DOI] [PubMed] [Google Scholar]
57.Marty MT; Baldwin AJ; Marklund EG; Hochberg GK; Benesch JL; Robinson CV, Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical chemistry 2015, 87 (8), 4370–4376. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Klotz IM, Ligand–receptor interactions: facts and fantasies. Quarterly reviews of biophysics 1985, 18 (3), 227–259. [DOI] [PubMed] [Google Scholar]
59.Lantz C; Zenaidee MA; Wei B; Hemminger Z; Ogorzalek Loo RR; Loo JA, ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry. Journal of proteome research 2021, 20 (4), 1928–1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Ulmer TS; Bax A; Cole NB; Nussbaum RL, Structure and dynamics of micelle-bound human α-synuclein. Journal of Biological Chemistry 2005, 280 (10), 9595–9603. [DOI] [PubMed] [Google Scholar]
61.Bush MF; Hall Z; Giles K; Hoyes J; Robinson CV; Ruotolo BT, Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Analytical chemistry 2010, 82 (22), 9557–9565. [DOI] [PubMed] [Google Scholar]
62.Picache JA; Rose BS; Balinski A; Leaptrot KL; Sherrod SD; May JC; McLean JA, Collision cross section compendium to annotate and predict multi-omic compound identities. Chemical science 2019, 10 (4), 983–993. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Ruotolo BT; Verbeck IV GF; Thomson LM; Woods AS; Gillig KJ; Russell DH, Distinguishing between phosphorylated and nonphosphorylated peptides with ion mobility− mass spectrometry. Journal of proteome research 2002, 1 (4), 303–306. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

JASMS SI 2023

NIHMS1977157-supplement-JASMS_SI_2023.pdf^{(1.7MB, pdf)}

[R1] 1.Johnson VE; Stewart W; Smith DH, Widespread tau and amyloid-beta pathology many years after a single traumatic brain injury in humans. Brain pathology 2012, 22 (2), 142–149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Díaz-Hernández M; Moreno-Herrero F; Gómez-Ramos P; Morán MA; Ferrer I; Baró AM; Avila J; Hernández F; Lucas JJ, Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain. Journal of Neuroscience 2004, 24 (42), 9361–9371. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Yamada M; Hayashi S; Tsuji S; Takahashi H, Involvement of the cerebral cortex and autonomic ganglia in Machado-Joseph disease. Acta neuropathologica 2001, 101 (2), 140–144. [DOI] [PubMed] [Google Scholar]

[R4] 4.Lautenschläger J; Stephens AD; Fusco G; Ströhl F; Curry N; Zacharopoulou M; Michel CH; Laine R; Nespovitaya N; Fantham M, C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction. Nature communications 2018, 9 (1), 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Bellani S; Sousa VL; Ronzitti G; Valtorta F; Meldolesi J; Chieregatti E, The regulation of synaptic function by α-synuclein. Communicative & Integrative Biology 2010, 3 (2), 106–109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Schweighauser M; Shi Y; Tarutani A; Kametani F; Murzin AG; Ghetti B; Matsubara T; Tomita T; Ando T; Hasegawa K, Structures of α-synuclein filaments from multiple system atrophy. Nature 2020, 1–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Li B; Ge P; Murray KA; Sheth P; Zhang M; Nair G; Sawaya MR; Shin WS; Boyer DR; Ye S, Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel. Nature communications 2018, 9 (1), 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Baba M; Nakajo S; Tu P-H; Tomita T; Nakaya K; Lee V; Trojanowski JQ; Iwatsubo T, Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. The American journal of pathology 1998, 152 (4), 879. [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Stephens AD; Zacharopoulou M; Moons R; Fusco G; Seetaloo N; Chiki A; Woodhams PJ; Mela I; Lashuel HA; Phillips JJ, Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity. Nature Communications 2020, 11 (1), 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Kang L; Moriarty GM; Woods LA; Ashcroft AE; Radford SE; Baum J, N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Science 2012, 21 (7), 911–917. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Bu B; Tong X; Li D; Hu Y; He W; Zhao C; Hu R; Li X; Shao Y; Liu C, N-terminal acetylation preserves α-synuclein from oligomerization by blocking intermolecular hydrogen bonds. ACS Chemical Neuroscience 2017, 8 (10), 2145–2151. [DOI] [PubMed] [Google Scholar]

[R12] 12.Sato H; Arawaka S; Hara S; Fukushima S; Koga K; Koyama S; Kato T, Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson’s disease. Journal of Neuroscience 2011, 31 (46), 16884–16894. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Choi TS; Lee J; Han JY; Jung BC; Wongkongkathep P; Loo JA; Lee MJ; Kim HI, Supramolecular modulation of structural polymorphism in pathogenic α-synuclein fibrils using copper (II) coordination. Angewandte Chemie International Edition 2018, 57 (12), 3099–3103. [DOI] [PubMed] [Google Scholar]

[R14] 14.Uversky VN; Li J; Fink AL, Metal-triggered structural transformations, aggregation, and fibrillation of human α-synuclein a possible molecular link between parkinson′ s disease and heavy metal exposure. Journal of Biological Chemistry 2001, 276 (47), 44284–44296. [DOI] [PubMed] [Google Scholar]

[R15] 15.Byrd EJ; Wilkinson M; Radford SE; Sobott F, Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein. Journal of the American Society for Mass Spectrometry 2023, 34 (3), 493–504. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Konijnenberg A; Ranica S; Narkiewicz J; Legname G; Grandori R; Sobott F; Natalello A, Opposite structural effects of epigallocatechin-3-gallate and dopamine binding to α-synuclein. Analytical chemistry 2016, 88 (17), 8468–8475. [DOI] [PubMed] [Google Scholar]

[R17] 17.Hong D-P; Fink AL; Uversky VN, Structural characteristics of α-synuclein oligomers stabilized by the flavonoid baicalein. Journal of molecular biology 2008, 383 (1), 214–223. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Prabhudesai S; Sinha S; Attar A; Kotagiri A; Fitzmaurice AG; Lakshmanan R; Ivanova MI; Loo JA; Klärner F-G; Schrader T, A novel “molecular tweezer” inhibitor of α-synuclein neurotoxicity in vitro and in vivo. Neurotherapeutics 2012, 9 (2), 464–476. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Bieschke J; Russ J; Friedrich RP; Ehrnhoefer DE; Wobst H; Neugebauer K; Wanker EE, EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proceedings of the National Academy of Sciences 2010, 107 (17), 7710–7715. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Xu M; Loa-Kum-Cheung W; Zhang H; Quinn RJ; Mellick GD, Identification of a new α-synuclein aggregation inhibitor via mass spectrometry based screening. ACS chemical neuroscience 2019, 10 (6), 2683–2691. [DOI] [PubMed] [Google Scholar]

[R21] 21.Acharya S; Safaie BM; Wongkongkathep P; Ivanova MI; Attar A; Klärner F-G; Schrader T; Loo JA; Bitan G; Lapidus LJ, Molecular basis for preventing α-synuclein aggregation by a molecular tweezer. Journal of Biological Chemistry 2014, 289 (15), 10727–10737. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Nshanian M; Lantz C; Wongkongkathep P; Schrader T; Klärner F-G; Blümke A; Despres C; Ehrmann M; Smet-Nocca C; Bitan G, Native top-down mass spectrometry and ion mobility spectrometry of the interaction of tau protein with a molecular tweezer assembly modulator. Journal of the American Society for Mass Spectrometry 2018, 30 (1), 16–23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Vöpel T; Bravo-Rodriguez K; Mittal S; Vachharajani S; Gnutt D; Sharma A; Steinhof A; Fatoba O; Ellrichmann G; Nshanian M, Inhibition of huntingtin exon-1 aggregation by the molecular tweezer CLR01. Journal of the American Chemical Society 2017, 139 (16), 5640–5643. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Malik R; Meng H; Wongkongkathep P; Corrales CI; Sepanj N; Atlasi RS; Klärner F-G; Schrader T; Spencer MJ; Loo JA, The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS. Journal of Biological Chemistry 2019, 294 (10), 3501–3513. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Sinha S; Lopes DH; Du Z; Pang ES; Shanmugam A; Lomakin A; Talbiersky P; Tennstaedt A; McDaniel K; Bakshi R, Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. Journal of the American Chemical Society 2011, 133 (42), 16958–16969. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Samanta N; Ruiz-Blanco YB; Fetahaj Z; Gnutt D; Lantz C; Loo JA; Sanchez-Garcia E; Ebbinghaus S, Superoxide dismutase 1 folding stability as a target for molecular tweezers in SOD1-related amyotrophic lateral sclerosis. ChemBioChem 2022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Leong SL; Pham CL; Galatis D; Fodero-Tavoletti MT; Perez K; Hill AF; Masters CL; Ali FE; Barnham KJ; Cappai R, Formation of dopamine-mediated α-synuclein-soluble oligomers requires methionine oxidation. Free Radical Biology and Medicine 2009, 46 (10), 1328–1337. [DOI] [PubMed] [Google Scholar]

[R28] 28.Sarafian TA; Ryan CM; Souda P; Masliah E; Kar UK; Vinters HV; Mathern GW; Faull KF; Whitelegge JP; Watson JB, Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein. PLoS One 2013, 8 (5), e63557. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Kellie JF; Higgs RE; Ryder JW; Major A; Beach TG; Adler CH; Merchant K; Knierman MD, Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson’s disease brain tissue by intact protein mass spectrometry. Scientific reports 2014, 4, 5797. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Dutta S; Hornung S; Taha HB; Biggs K; Siddique I; Chamoun LM; Shahpasand-Kroner H; Lantz C; Herrera-Vaquero M; Stefanova N, Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples. ACS Chemical Neuroscience 2023, 14 (7), 1238–1248. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Ly T; Julian RR, Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry. Journal of the American Society for Mass Spectrometry 2011, 19 (11), 1663–1672. [DOI] [PubMed] [Google Scholar]

[R32] 32.Wongkongkathep P; Han JY; Choi TS; Yin S; Kim HI; Loo JA, Native top-down mass spectrometry and ion mobility MS for characterizing the cobalt and manganese metal binding of α-synuclein protein. Journal of the American Society for Mass Spectrometry 2018, 29 (9), 1870–1880. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.Taha HB; Chawla E; Bitan G, IM-MS and ECD-MS/MS Provide Insight into Modulation of Amyloid Proteins Self-Assembly by Peptides and Small Molecules. Journal of the American Society for Mass Spectrometry 2023. [DOI] [PubMed] [Google Scholar]

[R34] 34.Haselmann KF; Jørgensen TJ; Budnik BA; Jensen F; Zubarev RA, Electron capture dissociation of weakly bound polypeptide polycationic complexes. Rapid communications in mass spectrometry 2002, 16 (24), 2260–2265. [DOI] [PubMed] [Google Scholar]

[R35] 35.Shi SD-H; Hemling ME; Carr SA; Horn DM; Lindh I; McLafferty FW, Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry. Analytical Chemistry 2001, 73 (1), 19–22. [DOI] [PubMed] [Google Scholar]

[R36] 36.Zhurov KO; Fornelli L; Wodrich MD; Laskay ÜA; Tsybin YO, Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chemical Society Reviews 2013, 42 (12), 5014–5030. [DOI] [PubMed] [Google Scholar]

[R37] 37.Zhou M; Lantz C; Brown KA; Ge Y; Paša-Tolić L; Loo JA; Lermyte F, Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry. Chemical science 2020, 11 (48), 12918–12936. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Comisarow MB; Marshall AG, Resolution-enhanced Fourier transform ion cyclotron resonance spectroscopy. The Journal of Chemical Physics 1975, 62 (1), 293–295. [Google Scholar]

[R39] 39.Li H; Wongkongkathep P; Van Orden SL; Ogorzalek Loo RR; Loo JA, Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment. Journal of the American Society for Mass Spectrometry 2014, 25 (12), 2060–2068. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Lermyte F; Everett J; Lam YP; Wootton CA; Brooks J; Barrow MP; Telling ND; Sadler PJ; O’Connor PB; Collingwood JF, Metal ion binding to the amyloid β monomer studied by native top-down FTICR mass spectrometry. Journal of the American Society for Mass Spectrometry 2019, 30 (10), 2123–2134. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Lanucara F; Holman SW; Gray CJ; Eyers CE, The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nature chemistry 2014, 6 (4), 281–294. [DOI] [PubMed] [Google Scholar]

[R42] 42.Shi H; Atlasevich N; Merenbloom SI; Clemmer DE, Solution dependence of the collisional activation of ubiquitin [M+ 7H] 7+ ions. Journal of the American Society for Mass Spectrometry 2014, 25 (12), 2000–2008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] 43.Dixit SM; Polasky DA; Ruotolo BT, Collision induced unfolding of isolated proteins in the gas phase: past, present, and future. Current opinion in chemical biology 2018, 42, 93–100. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R44] 44.Bernstein SL; Liu D; Wyttenbach T; Bowers MT; Lee JC; Gray HB; Winkler JR, α-Synuclein: Stable compact and extended monomeric structures and pH dependence of dimer formation. Journal of the American Society for Mass Spectrometry 2004, 15 (10), 1435–1443. [DOI] [PubMed] [Google Scholar]

[R45] 45.Jhingree JR; Beveridge R; Dickinson ER; Williams JP; Brown JM; Bellina B; Barran PE, Electron transfer with no dissociation ion mobility–mass spectrometry (ETnoD IM-MS). The effect of charge reduction on protein conformation. International Journal of Mass Spectrometry 2017, 413, 43–51. [Google Scholar]

[R46] 46.Williams JP; Brown JM; Campuzano I; Sadler PJ, Identifying drug metallation sites on peptides using electron transfer dissociation (ETD), collision induced dissociation (CID) and ion mobility-mass spectrometry (IM-MS). Chemical communications 2010, 46 (30), 5458–5460. [DOI] [PubMed] [Google Scholar]

[R47] 47.Campuzano ID; Lippens JL, Ion mobility in the pharmaceutical industry: an established biophysical technique or still niche? Current Opinion in Chemical Biology 2018, 42, 147–159. [DOI] [PubMed] [Google Scholar]

[R48] 48.Moons R; Konijnenberg A; Mensch C; Van Elzen R; Johannessen C; Maudsley S; Lambeir A-M; Sobott F, Metal ions shape α-synuclein. Scientific reports 2020, 10 (1), 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Liu Y; Carver JA; Calabrese AN; Pukala TL, Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 2014, 1844 (9), 1481–1485. [DOI] [PubMed] [Google Scholar]

[R50] 50.Moons R; van der Wekken-de Bruijne R; Maudsley S; Lemière F; Lambeir A-M; Sobott F, Effects of Detergent on α-Synuclein Structure. A Native MS-Ion Mobility Study. International Journal of Molecular Sciences 2020, 21 (21), 7884. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.Ponzini E; De Palma A; Cerboni L; Natalello A; Rossi R; Moons R; Konijnenberg A; Narkiewicz J; Legname G; Sobott F, Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure. Journal of Biological Chemistry 2019, 294 (14), 5657–5665. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R52] 52.Lantz C In Structural Analysis of Gas-Phase Phosphoproteins, American Society for Mass Spectrometry, Atlanta, GA, Atlanta, GA, 2019. [Google Scholar]

[R53] 53.Bernstein SL; Dupuis NF; Lazo ND; Wyttenbach T; Condron MM; Bitan G; Teplow DB; Shea J-E; Ruotolo BT; Robinson CV, Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nature chemistry 2009, 1 (4), 326–331. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R54] 54.Bleiholder C; Do TD; Wu C; Economou NJ; Bernstein SS; Buratto SK; Shea J-E; Bowers MT, Ion mobility spectrometry reveals the mechanism of amyloid formation of Aβ (25–35) and its modulation by inhibitors at the molecular level: epigallocatechin gallate and scyllo-inositol. Journal of the American Chemical Society 2013, 135 (45), 16926–16937. [DOI] [PubMed] [Google Scholar]

[R55] 55.Zheng X; Liu D; Klärner F-G; Schrader T; Bitan G; Bowers MT, Amyloid β-protein assembly: the effect of molecular tweezers CLR01 and CLR03. The Journal of Physical Chemistry B 2015, 119 (14), 4831–4841. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] 56.Talbiersky P; Bastkowski F; Klärner F-G; Schrader T, Molecular clip and tweezer introduce new mechanisms of enzyme inhibition. Journal of the American Chemical Society 2008, 130 (30), 9824–9828. [DOI] [PubMed] [Google Scholar]

[R57] 57.Marty MT; Baldwin AJ; Marklund EG; Hochberg GK; Benesch JL; Robinson CV, Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical chemistry 2015, 87 (8), 4370–4376. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R58] 58.Klotz IM, Ligand–receptor interactions: facts and fantasies. Quarterly reviews of biophysics 1985, 18 (3), 227–259. [DOI] [PubMed] [Google Scholar]

[R59] 59.Lantz C; Zenaidee MA; Wei B; Hemminger Z; Ogorzalek Loo RR; Loo JA, ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry. Journal of proteome research 2021, 20 (4), 1928–1935. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R60] 60.Ulmer TS; Bax A; Cole NB; Nussbaum RL, Structure and dynamics of micelle-bound human α-synuclein. Journal of Biological Chemistry 2005, 280 (10), 9595–9603. [DOI] [PubMed] [Google Scholar]

[R61] 61.Bush MF; Hall Z; Giles K; Hoyes J; Robinson CV; Ruotolo BT, Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Analytical chemistry 2010, 82 (22), 9557–9565. [DOI] [PubMed] [Google Scholar]

[R62] 62.Picache JA; Rose BS; Balinski A; Leaptrot KL; Sherrod SD; May JC; McLean JA, Collision cross section compendium to annotate and predict multi-omic compound identities. Chemical science 2019, 10 (4), 983–993. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R63] 63.Ruotolo BT; Verbeck IV GF; Thomson LM; Woods AS; Gillig KJ; Russell DH, Distinguishing between phosphorylated and nonphosphorylated peptides with ion mobility− mass spectrometry. Journal of proteome research 2002, 1 (4), 303–306. [DOI] [PubMed] [Google Scholar]

PERMALINK

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition

Carter Lantz

Jaybree Lopez

Andrew K Goring

Muhammad A Zenaidee

Karl Biggs

Julian P Whitelegge

Rachel R Ogorzalek Loo

Frank-Gerrit Klärner

Thomas Schrader

Gal Bitan

Joseph A Loo

Abstract

Introduction

Experimental