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Biological molecular machines enable chemical transformations, assembly, replication and motility, but most distinctively drive chemical systems out of-equilibrium to sustain life1,2. In such processes, nanometre-sized machines produce molecular energy carriers by driving endergonic equilibrium reactions. However, transforming the work performed by artificial nanomachines3–5 into chemical energy remains highly challenging. Here, we report a light-fuelled small-molecule ratchet capable of driving a coupled chemical equilibrium energetically uphill. By bridging two imine6–9 macrocycles with a molecular motor10,11, the machine forms crossings and consequently adopts several distinct topologies by either a thermal (temporary bond-dissociation) or photochemical (unidirectional rotation) pathway. While the former will relax the machine towards the global energetic minimum, the latter increases the number of crossings in the system above the equilibrium value. Our approach provides a blueprint for coupling continuous mechanical motion performed by a molecular machine with a chemical transformation to reach an out-of-equilibrium state.

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch‐based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non‐dissociative electron transfer as a reductive stimulus in the gas phase and probe the consecutive co‐conformational transitions of a donor‐acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. A comparison of collision cross section distributions for analogous closed‐shell and radical molecular ions sheds light on their respective formation energetics, while variations in their respective infrared absorption bands evidence changes in intramolecular organization as the foldamer becomes more compact. These differences are compatible with the advent of radical‐pairing interactions., Gas‐phase non‐dissociative electron transfer is used for charge reduction of a donor‐acceptor oligorotaxane foldamer (green). The consecutive co‐conformational transition is monitored using ion mobility spectrometry and infrared ion spectroscopy. Comparing the collision cross section distributions (blue) and infrared spectra (red) recorded for analogous closed‐shell and radical systems highlights differences that can be attributed to radical‐pairing interactions.

The fragmentation of benzylpyridinium "thermometer" ions is widely used to quantify the energetics of ions studied by mass spectrometry and other hyphenated techniques such as ion mobility. The reaction pathway leads to a benzylium cation with the release of a neutral pyridine. Using trapped ion mobility spectrometry, we noticed that the addition of acetonitrile, present in the electrosprayed solvent mixture, could occur on some electrophilic benzylium cations. This process results in the formation of adducts and in the appearance of a supplementary mobility peak. We here demonstrate that the addition takes place both in the electrospray source and inside the mobility analyzer, thereby evidencing possible outflow of solvent vapors downstream the instrument. By further characterizing the initial kinetics and the resulting equilibrium linked with the addition reaction, we presently discuss these as alternative probes to calibrate ion temperature in the framework of ion mobility.

Modern ion mobility instrumentation is typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during analysis. Here, we quantitatively assessed the internal heating experienced by ions during trapped ion mobility spectrometry (TIMS) experiments. To this end, the fragmentation yields of fragile benzylpyridinium "thermometer" ions were monitored during both the accumulation and analysis steps inside the TIMS tunnel. The corresponding fragmentation rate constants were translated into a vibrational effective temperature

Ion Mobility (IM) coupled to Mass Spectrometry (MS) has been used for several decades, bringing a fast separation dimension to the MS detection. IM-MS is a convenient tool for structural elucidation. The folding of macromolecules is often assessed with the support of computational chemistry. However, this strategy is strongly dependent on computational initial guesses. Here, we propose the analysis of the Collision Cross-Section (CCS) trends of synthetic homopolymers based on a fitting method which does not rely on computational chemistry a prioris of the three-dimensional structures. The CCS trends were evaluated as a function of the polymer chain length and the charge state. This method is also applicable to mobility trends. It leads to two parameters containing all information available through IM(-MS) measurements. One parameter can be interpreted as an apparent density. The second parameter is related to the shape of the ions and leads us to introduce the concept of trends with constant apparent density. Based on the two fitting parameters, a method for IM trend predictions is elaborated. Experimental deviations from the predictions facilitate detecting structural rearrangements and three-dimensional structure differences of the cationized polymer ions. This leads for instance to an easy identification and prediction of the presence of different polymer topologies in complex polymer mixtures. The classification of predicted trends could as well allow for software-assisted data processing. Finally, we suggest the link between the CCS trends of homopolymers and those obtained from (monodisperse) biomolecules to interpret potential folding differences during IM-MS studies.

The number of studies referring to the structural elucidation of intact biomolecular systems using mass spectrometry techniques has gradually increased in the post-2000s literature topics. As part of native mass spectrometry, this domain capitalizes on the kinetic trapping of physiological folds in view of probing solution-like conformational properties of isolated molecules or complexes after their electrospray transfer to the gas phase. Despite its efficiency for a wide array of analytes, this approach is expected to be pushed to its limits when considering highly dynamic systems or when dealing with nonideal operating conditions. To circumvent these limitations, we challenge the adequacy of an original strategy based on cross-linkers to improve the gas-phase stability of isolated proteins and ensure the preservation of folded conformations when measuring with strong transmission voltages, by spraying from denaturing solvents, or trapping for extended periods of time. Tested on cytochrome c, myoglobin, and β-lactoglobulin cross-linked using BS3, we validated the process as structurally nonintrusive in solution using far-ultraviolet circular dichroism and unraveled the preservation of folded conformations showing better resilience to denaturation on cross-linked species using ion mobility. The resulting collision cross sections were found in agreement with the native fold, and a preservation of the proteins' secondary and tertiary structures was evidenced using molecular dynamics simulations. Our results provide new insights concerning the fate of electro-sprayed cross-linked conformers in the gas phase, while constituting promising evidence for the validation of this technique as part of future structural mass spectrometry workflows.

Recent literature provides increasing samples of structural studies relying on ion mobility coupled to mass spectrometry in view of characterizing gas-phase conformation and energetics properties of biomolecular ions. A typical framework consists in experimentally monitoring the collisional cross sections for various experimental conditions and using them as references to select appropriate candidate structures issued from theoretical modeling. Although it has proved successful for structural assignment, this process is resource costly and lengthy, namely due to intricacies in the selection of appropriate input geometries. In the present work, we propose simplified methodologies dedicated to the systematic screening of ion mobility data acquired on systems built from repetitive subunits and detail their application to challenging artificial molecular switch systems. Capitalizing on coarse-grained design, we first demonstrate how the assimilation of subunits into adequately assembled building-blocks can be used for fast assignments of a system topology. Further focusing on topology-specific differential ion mobility trends, we show that the building-block assemblies can be fused into single fully convex solid figure models, i.e., sphere and cylinder, whose projected areas follow a two-parameter power formalism

Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract ᅟ.

Mass spectrometry (MS)-based proteomics is nowadays considered as a mainstream analytical tool for life sciences. This success is mainly to be attributed to the tremendous improvements in the core technology of mass spectrometry coupled with recent developments in bioinformatics and separation sciences. The level of information that is henceforth accessible through MS techniques is broad and encompasses the protein sequence as well as its quantity, modification(s), structure, and implication in macromolecular assemblies. The current chapter focuses on the existing gel-free MS-based quantitative approaches that are applicable in the field of animal sciences and summarizes their principal features together with their respective advantages and drawbacks. In this context, we will discuss both the label-free and label-based relative quantitation strategies as well as the existing alternatives for absolute quantitation. The specific case of posttranslational modifications, whose extensive characterization is becoming a crucial topic in MS-based proteome studies, is next addressed under the quantitative scope of phosphorylation and glycosylation. Lastly, other promising nonmainstream techniques are presented, emphasizing their potent use to solve major proteomics challenges of tomorrow.

State-of-the-art characterization of proteins using MS namely relies on fragmentation methods that allow exploring featured dissociative reaction pathways. These pathways are often initiated by a series of potentially informative mass-constant conformational changes that are nonetheless frequently overlooked by lack of adequate investigation techniques. In the present study, we propose a methodology to readily address both structural and energetic aspects of stereoisomerization reactions using ion mobility coupled with MS. To this end, a commercial spectrometer was used as a reactor comprising an energy resolved collisional activation step intended at promoting controlled conformational changes and a structural assignment step dedicated to the identification of the generated isomers. This identification relies on ion mobility and other on-line coupled techniques, namely an originally designed gas-phase H/D exchange experiment. We here apply this methodology to characterize the isomerization kinetics of capistruin, a 19-residue long lasso-folded peptide. We expect this approach to bring insights into the physical origin of global dissociation thresholds monitored in MS/MS experiments and to set a promising basis for quantitative investigations of the stability of different molecular folds.

At the interface between foldamers and mechanically interlocked molecules, oligorotaxanes exhibit a spring-like folded secondary structure with remarkable mechanical and physicochemical properties. Among these properties, the ability of oligorotaxanes to act as molecular switches through controlled modulations of their spatial extension over (un)folding dynamics is of particular interest. The present study aims to assess and further characterize this remarkable feature in the gas phase using mass spectrometry tools. In this context, we focused on the [4]5NPR+12 oligorotaxane molecule complexed with PF6– counterion and probed its co-conformational states as a function of the in-source-generated charge states. Data were interpreted in light of electronic secondary structure computations at the PM6 and DFT levels. Our results highlight two major co-conformational groups associated either with folded compact structures, notably stabilized by intramolecular π–π interactions and predominant for low charge state...

Hyperbranched and star shaped polymers have raised tremendous interest because of their unusual structural and photochemical properties, which provide them potent applications in various domains, namely in the biomedical field. In this context, the development of adequate tools aiming to probe particular three-dimensional features of such polymers is of crucial importance. In this present work, ion mobility coupled with mass spectrometry was used to experimentally derive structural information related to cationized linear and star shaped poly-ε-caprolactones as a function of their charge state and chain length. Two major conformations were observed and identified using theoretical modeling: (1) near spherical conformations whose sizes are invariant with the polymer topology for long and lightly charged chains and (2) elongated conformations whose sizes vary with the polymer topology for short and highly charged chains. These conformations were further confirmed by collisional activation experiments based on the ejection thresholds of the coordinated cations that vary according to the elongation amplitude of the polymer chains. Finally, a comparison between solution and gas-phase conformations highlights a compaction of the structure with a loss of specific chain arrangements during the ionization and desolvation steps of the electrospray process, fueling the long-time debated question related to the preservation of the analyte structure during the transfer into the mass spectrometer.

During their travel inside a traveling wave ion mobility cell (TW IMS), ions are susceptible to heating because of the presence of high intensity electric fields. Here, we report effective temperatures Teff,vib obtained at the injection and inside the mobility cell of a SYNAPT G2 HDMS spectrometer for different probe ions: benzylpyridinium ions and leucine enkephalin. Using standard parameter sets, we obtained a temperature of ~800 K at injection and 728 ± 2 K into the IMS cell for p-methoxybenzylpyridinium. We found that Teff,vib inside the cell was dependent on the separation parameters and on the nature of the analyte. While the mean energy of the Boltzmann distributions increases with ion size, the corresponding temperature decreases because of increasing numbers of vibrational normal modes. We also investigated conformational rearrangements of 7+ ions of cytochrome c and reveal isomerization of the most compact structure, therefore highlighting the effects of weak heating on the gas-phase structure of biologically relevant ions.

The dissociation of benzylpyridinium "thermometer" ions is widely used to calibrate the internal energy of ions produced in mass spectrometry. The fragmentation mechanism is usually believed to yield a benzylium cation, although recent studies suggest the possibility of a rearrangement leading to the tropylium isomer, which would compromise the accuracy of energy calibrations. In this study, we used IRMPD spectroscopy to probe the dissociation pathways of the p-(tert-butyl)benzylpyridinium ion. Our results show that the formation of both benzylium and tropylium products is feasible depending on the activation regime and on the reaction time scale. Varying the trapping delays in the hexapole gives insight into a rearrangement mechanism occurring through consecutive reactions with an isomerization from benzylium to tropylium. Our work provides experimental validations for the established calibration procedure and highlights the adequacy of IRMPD spectroscopy to qualitatively resolve gas-phase rearrangement kinetics. [GRAPHICS]

International audience; Traveling wave ion mobility spectrometers (TW IMS) operate at significantly higher fields than drift tube ion mobility spectrometers. Here we measured the fragmentation of the fragile p-methoxybenzylpyridinium ion inside the TW ion mobility cell of the first-generation SYNAPT HDMS spectrometer. The ion’s vibrational internal energy was quantified by a vibrational effective temperature Teff,vib, which is the mean temperature of the ions inside the cell that would result in the same fragmentation yield as observed experimentally. Significant fragmentation of the probe ion inside the TW IMS cell was detected, indicating that field heating of the ions takes place in TW IMS. For typical small molecule IMS conditions, Teff,vib = 555 ± 2 K. The variations of the effective temperature were studied as a function of the IMS parameters, and we found that Teff,vib decreases when the wave height decreases, when the pressure increases, or when the wave speed increases. The energy transfer efficiency of argon is higher than for He, N2 or CO2. Teff,vib being directly related to the ion speed inside the TW IMS, our results also provide new insight on the ion movement in TW IMS. We also discuss the influence of field heating of ions for calibration and structural studies in TW IMS.

Morsa Denis. Compte rendu de la réunion du samedi 13 mars 2004.. In: Revue belge de philologie et d'histoire, tome 82, fasc. 4, 2004. Histoire médiévale, moderne et contemporaine - Middeleeuwse. moderne en hedendaagse geschiedenis. pp. 1203-1209.

Urbanization of Belgium (1500-1800). Size, Hierarchy and Dynamics of the Towns. The urbanization of Belgium in the Modern Times is in no way an unknown territory. On the contrary, this field was recently the object of significant advances. However the research that has been carried out has not exhausted the subject. For example, an operational list of the Belgian communities regarded as towns between 1500 and 1800 is still lacking. And the urbanization process has not yet been examined from the angle of systemics. These observations led us in a first stage to draw a list of the Belgian towns of the modern period. Then this data base was used to understand the dynamics of the cities. Through the prism of the distribution of the towns according to their size, through rank-size adjustments, through a modelling of town generation and the use of allometric coefficients, one observes that the logic that presided over the organization of the urban network is completely altered in the XVIIIth century. From now on, the extension of the system no longer relies on the development of the more populated units (> 20,000 inhabitants) but, on the opposite, feeds on the growth of the towns situated in the intermediate to lower ranks of the hierarchy. This phenomenon can be tied to the process of « ruralization » of manufacturing taking place principally in the provinces of western and eastern Flanders. The analysis also reflects the insufficient integration of the Belgian space in the XVIth-XVIIIth centuries, period in which coexist sub-systems imperfectly interlocked. The urban outlook of Belgium in the modern period reflects the picture of a splintered country that renounced to organize itself around a metropolis of large dimensions. In short, the urbanization process of Belgium under the Ancien Régime owes much to economic and political factors., L'urbanisation de la Belgique au cours des Temps modernes ne constitue pas un thème inédit. Au contraire, le sujet a bénéficié récemment d'avancées significatives. Toutefois, les travaux qui se sont succédé sont loin d'avoir épuisé la matière. Par exemple, une liste opérationnelle des localités belges considérées comme villes entre 1500 et 1800 manque toujours à l'appel. D'autre part, le processus d'urbanisation n'a guère été examiné à l'aune de la systémique. Ces constats nous ont incité dans une première étape à dresser le répertoire des villes belges de l'ère moderne. Ensuite, on a exploité la base de données afin de saisir la dynamique des villes. A travers le prisme des distributions des villes selon leur taille, des ajustements rang-dimension, du modèle des générations de villes et des coefficients d'allométrie, on observe que la logique qui présidait à l'organisation du système urbain se modifie dans une mesure radicale au XVIIIe siècle. Désormais, l'extension du système ne procède plus de l'essor des unités les plus peuplées (> 20 000 habitants) mais au contraire s'alimente de la poussée des localités situées aux rangs intermédiaires et inférieurs de la hiérarchie. Le phénomène se rattache à la « ruralisation » de l'industrie dont principalement les provinces de Flandre orientale et occidentale font l'expérience. L'analyse reflète également l'intégration insuffisante de l'espace belge aux XVIe-XVIIIe siècles, au sein duquel coexistent des sous-systèmes imparfaitement emboîtés. La configuration urbaine de la Belgique à l'époque moderne renvoie l'image d'un pays éclaté qui a renoncé à se centraliser autour d'une métropole de grandes dimensions. Bref, le processus d'urbanisation de la Belgique sous l'Ancien Régime est largement tributaire des facteurs économiques et politiques., De ver stedelij king van België (1500-1800). Omvang, rangorde en dynamiek van de steden. De ver stedelij king van België in de moderne tijd is geen onontgonnen onderzoeksgebied. In de voorbije jaren is er juist op dat vlak heel wat vooruitgang geboekt. Toch kan men niet zeggen dat deze materie nu uitputtend behandeld is. Zo ontbreekt er nog altijd een handzame lijst van de plaatsen die men in de betreffende periode als steden kan beschouwen. Het verstedelijkingsproces is nauwelijks volgens een bepaald systeem onderzocht. Dat heeft er ons toe gebracht allereerst een lijst op te steilen van de Belgische steden in de moderne tijd. Daarna werd de gegevensbank gebruikt om de dynamiek van de steden te vatten. Via een opdeling van de steden naar formaat en rangorde, naar het generatiemodel en allometrische coefficienten, kan men vaststellen dat de logica van de organisatie van het stedelijk systeem op een radikale manier gewijzigd wordt in de XVIIIde eeuw. Van dan af is de uitbreiding geen gevolg meer van de opgang van de meest bevolkte eenheden (meer dan 20 000 inwoners), maar van plaatsen die een lagere of middenplaats in de hiérarchie bekleden. Dat fenomeen is verbonden met de « ruralisatie » van de industrie die vooral zichtbaar is in de provincies Oosten West-Vlaanderen. Uit de analyse blijkt ook de onvoldoende integratiegraad van de Belgische ruimte in de XVI-XVIIIde eeuw, waarin subsystemen naast elkaar blijven bestaan zonder wederzijdse aanpassing. Het beeld van verstedelijkt België in de moderne periode toont een verdeeld land dat zich niet gericht heeft op een enkele grote stedelijke kern. Kortom, het urbanisatieproces van België tijdens het Ancien Régime hangt nauw samen met economische en politieke factoren., Morsa Denis. L'urbanisation de la Belgique (1500-1800). Taille, hiérarchie et dynamique des villes.. In: Revue du Nord, tome 79, n°320-321, Avril-septembre 1997. pp. 303-330.

Morsa Denis. Une croissance manquée : Huy (1600-1800). Quelques hypothèses sur un effondrement économique. In: Revue belge de philologie et d'histoire, tome 59, fasc. 2, 1981. Histoire médiévale, moderne et contemporaine — Middeleeuwse, moderne en hedendaagse geschiedenis. pp. 325-381.

Denis Morsa. Small towns in the principality of Liège at the end of the 18th century. First approaches. This analysis of small towns in the principality of Liège at the end of the 18th century is an attempt to contribute to the study of the problems of urbanization in Western Europe. It is divided in several steps: the definition of criteria to distinguish small towns from areas and villages inhabited by country people, the evaluation of the popupation of the entities described, the use of precise statistical processes to check the correspondence of the Liège urban structure to some distribution types or normative hierarchies. This approach throws a new light on two essential elements of the rban net in the region of Liège: immaturity and general lack of balance., Denis Morsa. Les petites villes dans la principauté de Liège à la fin du XVnie diècle. Premières approches. En étudiant les petites villes dans la principauté de Liège à la fin du XVIIIe siècle, il s'agit d'apporter une contribution à la problématique de l'urbanisation en Europe occidentale. L'analyse progresse en plusieurs étapes : mise au point d'une critériologie pour distinguer les petites villes des agglomérations et communautés d'habitants du plat-pays ; évaluation des effectifs de la population des localités prises en compte ; mise en oeuvre de procédés statistiques explicitement définis afin de tester le degré de conformité de l'armature urbaine liégeoise par rapport à certaines distributions types ou à des hiérarchies normatives. Cette démarche met en lumière deux composantes essentielles du réseau urbain liégeois : immaturité et déséquilibre d'ensemble., Morsa Denis. Les petites villes dans la principauté de Liège à la fin du XVIIIe siècle. Premières approches. In: Histoire & Mesure, 1987 volume 2 - n°2. Varia. pp. 73-91.

Morsa Denis. Salaire et salariat dans les économies préindustrielles (XVIe-XVIIIe siècle). Quelques considérations critiques. In: Revue belge de philologie et d'histoire, tome 65, fasc. 4, 1987. Histoire - Geschiedenis. pp. 751-784.

Salamagne Alain. Philippe II de Croÿ et la fortification des villes de Hainaut : Avesnes, Bouchain, Le Quesnoy, trois chantiers renaissants de la décennie 1530. In: Revue belge de philologie et d'histoire, tome 89, fasc. 2, 2011. Villes et villages : organisation et représentation de l’espace. Mélanges offerts à Jean-Marie Duvosquel à l’occasion de son soixante-cinquième anniversaire et publiés par Alain Dierkens, Christophe Loir, Denis Morsa, Guy Vanthemsche. pp. 685-700.