Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures.

Di-tyrosine is one of the major protein cross-links involved in a large number of neurodegenerative or ageing-related diseases. Recently, no less than four different di-tyrosine bridge isomers have been highlighted while only two structures are characterized at the moment in the literature. In this study, the four dimers were produced by radiolytical-induced oxidation. Although the abundance of these additional dimers precluded the use of NMR or other structural characterization methods, we propose a new methodology combining UPLC-MS analysis, specific deuterium labelling and isotopic (H/D) exchanges with the solvent. Thus, we were able to identify three different covalent cross-links and propose different new original di-tyrosine structures based on double Michael additions, leading to tetracyclic products. Absorption and fluorescence characterizations of the four species were performed and consolidate our proposal.

Chemical Processes Involving 18-Crown-6-Ether in Activated Noncovalent Complexes with Protonated Peptides.

These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.

Helical γ-Peptide Foldamers as Dual Inhibitors of Amyloid-ß Peptide and Islet Amyloid Polypeptide Oligomerization and Fibrillization.

Type 2 diabetes (T2D) and Alzheimer's disease (AD) belong to the 10 deadliest diseases and are sorely lacking in effective treatments. Both pathologies are part of the degenerative disorders named amyloidoses, which involve the misfolding and the aggregation of amyloid peptides, hIAPP for T2D and Aß1-42 for AD. While hIAPP and Aß1-42 inhibitors have been essentially designed to target ß-sheet-rich structures composing the toxic amyloid oligomers and fibrils of these peptides, the strategy aiming at trapping the non-toxic monomers in their helical native conformation has been rarely explored. We report herein the first example of helical foldamers as dual inhibitors of hIAPP and Aß1-42 aggregation and able to preserve the monomeric species of both amyloid peptides. A foldamer composed of 4-amino(methyl)-1,3-thiazole-5-carboxylic acid (ATC) units, adopting a 9-helix structure reminiscent of 310 helix, was remarkable as demonstrated by biophysical assays combining thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis and mass spectrometry.

Evidence of conformational landscape alteration and macromolecular complex formation in the early stages of in vitro human prion protein oxidation.

Oxidative stress is proposed to be one of the major causes of neurodegenerative diseases. Cellular prion protein (PrP) oxidation has been widely studied using chemical reagents such as hydrogen peroxide. However, the experimental conditions used do not faithfully reflect the physiological environment of the cell. With the goal to explore the conformational landscape of PrP under oxidative stress, we conducted a set of experiments combining the careful control of the nature and the amount of ROS produced by a60Co γ-irradiation source. Characterization of the resulting protein species was achieved using a set of analytical techniques. Under our experimental condition hydroxyl radical are the main reactive species produced. The most important findings are i) the formation of molecular assemblies under oxidative stress, ii) the detection of a majority of unmodified monomer mixed with oxidized monomers in these molecular assemblies at low hydroxyl radical concentration, iii) the absence of significant oxidation on the monomer fraction after irradiation. Molecular assemblies are produced in small amounts and were shown to be an octamer. These results suggest either i) an active recruitment of intact monomers by molecular assemblies' oxidized monomers then inducing a structural change of their intact counterparts or ii) an intrinsic capability of intact monomer conformers to spontaneously associate to form stable molecular assemblies when oxidized monomers are present. Finally, abundances of the intact monomer conformers after irradiation were modified. This suggests that monomers of the molecular assemblies exchange structural information with intact irradiated monomer. All these results shed a new light on structural exchange information between PrP monomers under oxidative stress.

Evidence for different in vitro oligomerization behaviors of synthetic hIAPP obtained from different sources.

Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24 h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process. Graphical abstract.

Improved Infrared Spectra Prediction by DFT from a New Experimental Database.

This work aims to improve the computation of infrared spectra of gas-phase cations using DFT methods. Experimental infrared multiple photon dissociation (IRMPD) spectra for ten Zn and Ru organometallic complexes have been used to provide reference data for 64 vibrational modes in the 900-2000 cm-1 range. The accuracy of the IR vibrational frequencies predicted for these bands has been assessed over five DFT functionals and three basis sets. The functionals include the popular B3LYP and M06-2X hybrids and the range-separated hybrids (RSH) CAM-B3LYP, LC-BLYP, and ωB97X-D. B3LYP gives the best mean absolute error (MAE) and root-mean-square error (RMSE) values of 7.1 and 9.6 cm-1 , whilst the best RSH functional, ωB97X-D, gives 12.8 and 16.6 cm-1 , respectively. Using linear correlations instead of scaling factors improves the prediction accuracy significantly for all functionals. Experimental and computed spectra for a single complex can show significant differences even when the molecular structure is calculated correctly, and a means of defining confidence limits for any given computed structure is also provided.

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Combining gas phase electron capture and IRMPD action spectroscopy to probe the electronic structure of a metastable reduced organometallic complex containing a non-innocent ligand.

Combining electron capture dissociation mass spectrometry and infrared multiple photon dissociation action spectroscopy allows the formation, selection and characterisation of reduced metal complexes containing non-innocent ligands. Zinc complexes containing diazafluorenone ligands have been studied and the localisation of the single electron on the metal atom in the mono-ligated complex has been demonstrated.

Proteomics Methodologies: The Search of Protein Biomarkers Using Microfluidic Systems Coupled to Mass Spectrometry.

Protein biomarkers have been the subject of intensive studies as a target for disease diagnostics and monitoring. Indeed, biomarkers have been extensively used for personalized medicine. In biological samples, these biomarkers are most often present in low concentrations masked by a biologically complex proteome (e.g., blood) making their detection difficult. This complexity is further increased by the needs to detect proteoforms and proteome complexity such as the dynamic range of compound concentrations. The development of techniques that simultaneously pre-concentrate and identify low-abundance biomarkers in these proteomes constitutes an avant-garde approach to the early detection of pathologies. Chromatographic-based methods are widely used for protein separation, but these methods are not adapted for biomarker discovery, as they require complex sample handling due to the low biomarker concentration. Therefore, microfluidics devices have emerged as a technology to overcome these shortcomings. In terms of detection, mass spectrometry (MS) is the standard analytical tool given its high sensitivity and specificity. However, for MS, the biomarker must be introduced as pure as possible in order to avoid chemical noise and improve sensitivity. As a result, microfluidics coupled with MS has become increasingly popular in the field of biomarker discovery. This review will show the different approaches to protein enrichment using miniaturized devices and the importance of their coupling with MS.

Dereplication of Acetogenins from Annona muricata by Combining Tandem Mass Spectrometry after Lithium and Copper Postcolumn Cationization and Molecular Networks.

Annonaceous acetogenins are natural products held responsible for atypical Parkinsonism due to chronic consumption in traditional medicine or as food, leading to the development of analytical strategies for their complete chemical characterization in complex mixtures. Characterization by tandem mass spectrometry (MS/MS) of acetogenins using collision-induced dissociation from lithium adducts provides additional structural information compared to protonated or sodiated species such as ketone location on the acetogenin backbone. However, very low intensity diagnostic ions together with the lack of extensive structural information regarding position of OH and THF substituents limit this approach. Copper adducts led to diagnostic fragment ions that allow us to identify the position of oxygen rings and hydroxyl substituents. Fragmentation rules were established on the basis of acetogenin standards allowing the identification of 45 over the 77 analogues observed in an extract of Annona muricata by LC-MS/MS using postcolumn infusion of copper sulfate (CuSO4) solution. Molecular networks that were generated thanks to specific fragmentations obtained with copper led to the distinction of THF ring position or to the identification of hydroxylated lactone, for instance.

Oxidative radicals (HO• or N3•) induce several di-tyrosine bridge isomers at the protein scale.

Among protein oxidative damages, di-tyrosine bridges formation has been evidenced in many neuropathological diseases. Combining oxidative radical production by gamma radiolysis with very performant chromatographic separation coupled to mass spectrometry detection, we brought into light new insights of tyrosine dimerization. Hydroxyl and azide radical tyrosine oxidation leading to di-tyrosine bridges formation was studied for different biological compounds: a full-length protein (Δ25-centrin 2), a five amino acid peptide (KTSLY) and free tyrosine. We highlighted that both radicals generate high proportion of dimers even for low doses. Surprisingly, no less than five different di-tyrosine isomers were evidenced for the protein and the peptide. For tyrosine alone, at least four distinct dimers were evidenced. These results raise some questions about their respective role in vivo and hence their relative toxicity. Also, as di-tyrosine is often used as a biomarker, a better knowledge of the type of dimer detected in vivo is now required.

Conformation assessment of therapeutic monoclonal antibodies by SEC-MS: Unravelling analytical biases for application to quality control.

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection.

Prions are known to be involved in neurodegenerative pathologies such as Creutzfeld-Jakob disease. Current models point to a molecular event which rely on a transmissible structural change that leads to the production of ß-sheet-rich prion conformer (PrPSc). PrPSc itself has the capability to trigger the structural rearrangement of the ubiquitously present prion (PrPc) substrate in a self-perpetuating cascade. In this article, we demonstrate that recombinant PrPc exists in a conformational equilibrium. The conformers' abundances were shown to be dependent on PrPc concentration through the formation of transient multimers leading to conformational selection. The study of PrPc mutants that follow dedicated oligomerization pathways demonstrated that the conformers' relative abundances are modified, thus reinforcing the assertion that the nature of conformers' interactions orient the oligomerization pathways. Further this result can be viewed as the "signature" of an aborted oligomerization process. This discovery sheds a new light on the possible origin of prion protein diseases, namely that a change in prion protein structure could be transmitted through the formation of transient multimers having different conformer compositions. This could explain the selection of a transient multimeric type that could be viewed as the precursor of PrPSc responsible for structural information transmission, and strain apparition.

Structure of electron-capture dissociation fragments from charge-tagged peptides probed by tunable infrared multiple photon dissociation.

In this study, we propose the first spectroscopic structural characterization of c-type ions produced by ECD of a peptide. The structure of c-type ions formed by electron capture dissociation and the overall mechanism leading to their formation are still a question of debate. Depending on the mechanism, c-type ions have been proposed to have either an enol-imine structure (-C(OH)NH) or an amide one (-C(O)-NH2). Since these ions are isomeric, mass spectrometry only cannot discriminate between them, but infrared spectroscopy can bring experimental evidence and help determine which scheme is operative. Using the coupling between a tunable free electron laser and a FT-ICR mass spectrometer, we show that c-type ions have an amide structure, characterized by an IR signature of the C=O stretch at 1731 cm(-1). This result is particularly interesting from the perspective of the elucidation of the ECD mechanism.

[Nepsilon-(gamma-glutamyl) lysine] as a potential biomarker in neurological diseases: new detection method and fragmentation pathways.

Protein aggregates are characteristic of a number of diseases of the central nervous system such as diseases of polyQ expansion. Covalent bonds formed by the action of transglutaminase are thought to participate in the stabilization of these aggregates. Transglutaminase catalyzes the formation of cross-links between the side chains of glutaminyl and lysyl residues of polypeptides. Identification of the isodipeptide N(epsilon)-(gamma-glutamyl) lysine (iEK) in terminal proteolytic digests of neuronal aggregates would demonstrate participation of transglutaminase in neurological diseases. In order to identify and quantify the iEK present in the brain of patients with neurological disease, a method combining liquid chromatography and multistep mass spectrometry was developed. Because isobaric peptides of iEK could be present in the digest of aggregated proteins, the choice of fragment diagnostic ions was crucial. These ions were identified by mass spectrometry on sodiated iEK, which was derivatized on the carboxylic functions and terminal amines in order to improve sensitivity. Deuterated molecules as well as (13)C(6)- and (15)N(2)-isotopomers were used to derive filiations in the multistep fragmentations. The main fragmentation patterns have been identified, so that two ions (m/z 396 [MH - 56-42 u](+) and 350 [MH - 56-88 u](+)) are shown to be adequate markers for quantitation experiments. In order to gain a better understanding of the fragmentation processes, detailed quantum chemical calculations have been performed at levels which are expected to provide good accuracy. A thorough study has been carried out with a reduced model in which only the 'active' part of the molecule is retained. This allowed obtaining full mechanistic details on the pathways leading to a number of observed fragments. In particular, it has been shown that losses of 87 and 88 u from A(+) = [MH - 56 u](+) are competitive. Computations on the entire derivatized isodipeptide have been used to validate the use of the smaller model in order to obtain reliable energetics and mechanisms.

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides.

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups.

Size Exclusion Chromatography-Ion Mobility-Mass Spectrometry Coupling: a Step Toward Structural Biology.

Noncovalent interactions are essential for the structural organization of biomacromolecules in cells. For this reason, the study of the biophysical, dynamic, and architectural interactions among biomacromolecules is essential. Since mass spectrometry requires compatible solutions while preserving the noncovalent bonding network, we envisioned that size exclusion chromatography coupled with ion mobility and mass spectrometry would be a valuable technique to desalt the initial sample and provide solution and gas-phase structural information in a single stage experiment. Such coupling allowed obtaining information on solution protein complex composition with SEC separation and on authenticity and purity with IMS-MS. Our study demonstrated that such coupling is compatible, useful, as well as suitable for a routine analysis, in pharmaceutical industry, for example. Mobility data were reliable and injected standards allowed calibrating the collision cross-section scale. Graphical Abstract ᅟ.

Monitoring Conformational Landscape of Ovine Prion Protein Monomer Using Ion Mobility Coupled to Mass Spectrometry.

Prion protein is involved in deadly neurodegenerative diseases. Its pathogenicity is linked to its structural conversion (α-helix to ß-strand transition). However, recent studies suggest that prion protein can follow a plurality of conversion pathways, which hints towards different conformers that might coexist in solution. To gain insights on the plasticity of the ovine prion protein (PrP) monomer, wild type (A136, R154, Q171), mutants and deletions of ARQ were studied by traveling wave ion mobility experiments coupled to mass spectrometry. In order to perform the analysis of a large body of data sets, we designed and evaluated the performance of a processing pipeline based on Driftscope peak detection and a homemade script for automated peak assignment, annotation, and quantification on specific multiply charged protein data. Using this approach, we showed that in the gas phase, PrPs are represented by at least three conformer families differing in both charge state distribution and collisional cross-section, in agreement with the work of Hilton et al. (2010). We also showed that this plasticity is borne both by the N- and C-terminal domains. Effect of protein concentration, pH and temperature were also assessed, showing that (1) pH does not affect conformer distributions, (2) protein concentration modifies the conformational landscape of one mutant (I208M) only, and (3) heating leads to other unfolded species and to a modification of the conformer intensity ratios. Graphical Abstract ᅟ.

A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry.

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.