The development and commercialization of cyclic ion mobility spectrometry (cIMS) instruments has considerably expanded the level of structural details that can be accessed for gaseous ions. Commercial cIMS instruments rely on travelling wave ion mobility spectrometry (TWIMS), a technique that requires calibration of the measured arrival times to enable the determination of collisional cross sections (CCS). Most calibration strategies use peptide mixtures that span a CCS range of approximately 100 – 400 Å2, which can introduce significant inaccuracies when extrapolating to larger ion structures. In addition, TWIMS calibration remains a tedious data processing step that would greatly benefit from automation. Here, we introduce polycIMS, a command-line tool that automates cIMS calibration and generates charge-independent calibration curves covering a broad CCS range from 100 – 1500 Ų, within just 3 minutes on a standard laptop. Calibrated CCS can be determined in He or N 2 ( TW CCS N2→He , TW CCS N2→N2 ) using inexpensive, commercially-available polymer standards and are validated against reported values for more than 50 reference ions. Furthermore, the derived calibration parameters are largely insensitive to collisional activation, making it possible to calibrate multistage cIMS workflows such as slice collision induced unfolding (CIU n ). We anticipate that the simplicity and accessibility of polycIMS will encourage its adoption as a new standard for cIMS calibration.

Abstract Understanding and quantifying activation parameters associated with the gas-phase isomerization kinetics of stereoisomeric ions, such as Z- and E-azobenzene ions, remains a key challenge in modern mass spectrometry. Here, we explore collisional activation as a fast and efficient method to probe isomerization kinetics on QToF instruments equipped with traveling wave-ion mobility spectrometry (TWIMS), such as the Waters Synapt platform. Using sequence-defined peptoids bearing an azobenzene chromophore, we investigate the collision-induced isomerization (CII) of Z-isomer ions into stable E-isomer ions within the Trap Cell–IMS configuration. The extent of Z → E conversion is monitored as a function of the activation voltage (Trap CV), allowing the extraction of kinetic constants based on the known residence time of ions in the Trap Cell. By integrating previously determined gas-phase activation parameters (ΔH‡ and ΔS‡) into a modified Eyring framework, we derived effective temperatures (Teff) that characterize the internal energy distribution of activated ions. The dependence of Teff on Trap CV yields two critical parameters: α, describing the kinetic-to-internal energy conversion efficiency, and T0, the pre-activation ion population temperature. The measured α values are of the same order as those previously derived for protein ions. However, T0 values are found to be system-dependent, which is detrimental for establishing a universal temperature calibration. Ongoing work focuses on expanding the switchable molecule library to map α and T0 across diverse molecular architectures and decipher at the molecular level the origin of these two parameters.

The rational design of hybrid nanomaterials with precisely controlled properties remains a central challenge in materials science. While atomically precise gold nanoclusters (Au-NCs) offer molecule-like control over a metallic core, tuning their optoelectronic behavior via surface engineering is often empirically driven. Here, we establish a design principle by demonstrating that the conformational landscape of intrinsically disordered proteins (IDP) can be used as a programmable scaffold to rationally modulate the photophysical properties of a covalently bound Au-NC. We synthesized a series of bioconjugates between Au 25 nanoclusters and bioengineered IDPs containing a variable number of cysteine anchoring points. A combination of mass spectrometry, small-angle X-ray scattering, and modeling on the conjugates indicates that increasing the number of covalent anchors systematically restricts the conformational ensemble, inducing a progressively more compact protein shell around nanoclusters. This structural rigidification at the interface directly translates into a 15-fold enhancement of the Au-NC near-infrared photoluminescence and a six-fold increase in its average lifetime. Our findings demonstrate that the conformational plasticity of IDPs and the capacity to engineer them can be harnessed as a molecular tuning knob, moving to a new regime of programmable soft-matter control over the properties of quantum-confined nanomaterials for tailored biotechnological applications.

Storing solar energy in chemical bonds through the reversible isomerization of UV-vis absorbing molecules offers a promising approach to energy storage. These molecules form high-energy photoisomers, which can store energy if kinetically protected by a significant activation barrier against spontaneous thermal back-isomerization. In this study, we compare the back-isomerization kinetic parameters (ΔH‡ and ΔS‡) of model azobenzene-based photoswitches in solution with those obtained in the gas phase using an original tandem ion mobility mass spectrometer. Our findings show that the activation enthalpy is well-reproduced from the solution phase to the gas phase, whereas the activation entropy is significantly affected by the absence of solvent, revealing further different relaxation mechanisms.

Understanding how structural and optical properties of metallic nanoclusters can be tuned by proteins is crucial for the use of these hybrid molecules in biomedical applications. The interaction of proteins with ultrasmall, atomically-precise gold nanoclusters (Au-NCs) has been mainly investigated in the context of structured proteins, while their behavior with intrinsically disordered proteins (IDPs) remains unexplored. This work examines the structural and optical properties of Au-NCs interacting with bioengineered IDPs containing up to three cysteines. We show that, by exploiting the conformational flexibility of cysteine-containing IDPs, we can anchor proteins to Au-NCs in a position-specific manner, leading to new bioconjugates with properties that differ from those of the individual components. We observed an up to 15-fold photoluminescence enhancement depending on the number of cysteines anchored. By combining mass spectrometry, small-angle X-ray scattering (SAXS), and computational modelling, the ensemble structures of nine bioconjugates with different stoichiometries were elucidated, indicating their overall compactness. Our results suggest that the interface between these atomically-precise species and the conformationally fluctuating protein is responsible for the optical properties of these nanobioconjugates. This research improves our understanding of Au-NC– protein interactions, paving the way to novel nano-molecular hybrid conjugates with tunable properties for bioimaging and therapeutic applications. ### Competing Interest Statement The authors have declared no competing interest. * NC : nanoclusters IDP : intrinsically disordered protein NIR : near-infrared pMBA : para-mercaptobenzoic acid cys : cysteine R g : gyration radius PAGE : Polyacrylamide Gel Electrophoresis MS : Mass Spectrometry MW : Molecular Weight PL : photoluminescence SAXS : Small-Angle X-ray Scattering SEC : Size-Exclusion Chromatography EOM : Ensemble Optimization Method ICT : intramolecular charge transfer BSA : Bovine Serum Albumin. Agence Nationale de la Recherche, ANR-22-CE29-0022, ANR-20-CE92-0039-01 Deutsche Forschungsgemeinschaft, DFG RE 1203/38-1

Red luminophores displaying large Stokes shift and high-quantum yields are obtained when gold salts are reacted with proteins under strongly alkaline conditions. Although bovine serum albumin (BSA) has mainly been used as a protein template, other attempts to prepare red luminophores have been proposed using other proteins. Here, we report on the structural characterization and nonlinear optical properties of insulin-gold conjugates. Such conjugates display strong luminescence at ∼670 nm with quantum yields that reach 5.4%. They also display long luminescence lifetimes allowing efficient reactive oxygen species generation, with a quantum yield of 1O2 generation reaching 13%. In addition, they exhibit remarkable nonlinear optical properties and in particular a strong two-photon excited fluorescence (TPEF) cross section in the range of 800-1100 nm. By combining experimental studies and time-dependent density functional theory simulations (TD-DFT), we show the formation of insulin-Au(III) conjugates. The interaction of Au(III) ions with the aromatic rings of tyrosine induces charge transfer-like excitation in the visible range. Experimental investigations, together with molecular dynamics simulations of insulin and calculations of electronic properties in a model system, are performed to explore the origin of optical features and the structure-optical property relationship, leading the way to new concepts for nonlinear optics using protein-Au(III) conjugates.

Protein protected metal nanoclusters (NCs) represent a new class of highly photoluminescent nanomaterials that have wide applications. Suitable reaction conditions combining protein and metal precursors can produce a vast range of different NC sizes. The number of metal atoms per protein can be determined by mass spectrometry (MS). MS coupled with matrix-assisted laser desorption ionization (MALDI) presents a number of advantages such as detection with high sensitivity of nanoclusters with high molecular weights. Although many protein-protected metal nanoclusters have been characterized by MALDI-MS, a large dispersion in the number of metal atoms have been reported mainly due to sample preparation. In this work, we optimized the protocols for negative and positive ion detection mode as a general MALDI-MS sample preparation method for protein protected metal nanoclusters (bovine serum albumin and lysozyme and with gold and silver). Negative and positive ion mode detection was compared, showing that negative ion mode detection in MALDI-MS can also be used with acidic matrices. Obvious matrix effects on ion signals and peak positions by MALDI-MS was observed. The metal atom number of metal nanoclusters embedded in proteins is different depending on the MALDI matrix most probably due to fragmentation. The matrix effects give a warning for more serious consideration on MALDI-MS measurement and spectra analysis of nanoclusters.

The resistance of gram-negative bacteria to silver ions is mediated by a silver efflux pump, which mainly relies on a tripartite efflux complex SilCBA, a metallochaperone SilF and an intrinsically disordered protein SilE. However, the precise mechanism by which silver ions are extruded from the cell and the different roles of SilB, SilF, and SilE remain poorly understood. To address these questions, we employed nuclear magnetic resonance and mass spectrometry to investigate the interplay between these proteins. We first solved the solution structures of SilF in its free and Ag+-bound forms, and we demonstrated that SilB exhibits two silver binding sites in its N and C termini. Conversely to the homologous Cus system, we determined that SilF and SilB interact without the presence of silver ions and that the rate of silver dissociation is eight times faster when SilF is bound to SilB, indicating the formation of a SilF-Ag-SilB intermediate complex. Finally, we have shown that SilE does not bind to either SilF or SilB, regardless of the presence or absence of silver ions, further corroborating that it merely acts as a regulator that prevents the cell from being overloaded with silver. Collectively, we have provided further insights into protein interactions within the sil system that contribute to bacterial resistance to silver ions.

Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

We developed a new environment-sensing device based on the opto-ionic-electronic phenomena of an octahedral molybdenum metal (Mo 6 ) cluster. When the Mo 6 cluster is electrochemically deposited on a transparent electrode in an organic solvent containing a trace amount of water, the water permeates the deposited film. During the process, some ligand species that stabilize the frame structure of the Mo 6 cluster are substituted with hydroxyl groups, and the negatively charged frame structure of the Mo 6 cluster unit is stabilized by hydronium counterions. As a result, the transparent film of the Mo 6 cluster fabricated by this method exhibits ionic-electronic mixed conduction of the hydronium ion. The ionic conduction greatly changes depending on the temperature and humidity in the atmosphere, and the electrical conductivity greatly changes depending on the wavelength and intensity of the irradiated light. These unique multisensing properties present new possibilities for environmental sensing applications.

Many sophisticated approaches for analyzing properties of chiral matter have been developed in recent years. But in general, the available chiroptical methods are limited to either solvated or small gaseous molecules. Studying the chirality of large biopolymers in the gas phase, including aspects of the secondary structure, becomes accessible by combining the electrospray ionization technique with chiroptical detection protocols. Here, laser-induced photodetachment from gramicidin anions, a peptide consisting of 15 amino acids has been investigated. The angular distribution of photoelectrons is demonstrated to be sensitive to the substitution of protons by cesium ions, which is accompanied by a conformational change. The photoelectron circular dichroism (PECD) is -0.5% for bare gramicidin, whereas gramicidin with several Cs+ ions attached exhibits a PECD of +0.5%. The results are complemented and supported by ion mobility studies. The presented approach offers the prospect of studying chirality and the secondary structure of various biopolymers.

The dynamics and thermodynamics of structural changes in isolated glu-fibrinopeptide B (GluFib) were investigated by tandem ion mobility spectrometry (IMS). Doubly protonated GluFib2+ ions were first selected by IMS and then stored for a controlled duration in a thermalized ion trap. Temperature-induced conformational changes were finally monitored by IMS as a function of trapping time. Based on this procedure, isomerization rates and equilibrium populations of the different conformers were determined as a function of temperature. We demonstrate that the measured thermodynamic quantities can be directly compared to simulated observables from ensemble molecular modeling based on appropriate order parameters. We obtained good qualitative agreement with replica-exchange molecular dynamics simulations based on the AMOEBA force field and processed using the weighted histogram analysis method. This suggests that the balance between Coulomb repulsion and optimal charge solvation is the main source of the observed conformational bistability. Our results emphasize the differences between the kinetically driven quasi-equilibrium distributions obtained after collisional activation and the thermodynamically driven distributions from the present equilibrium experiments due to entropic effects. As a consequence, our measurements not only allow straightforward determination of Arrhenius activation energies but also yield the relative enthalpy and entropy changes associated to a structural transition.

Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.

Edge-bridged halide tantalum clusters based on the {Ta6Br12}4+ core have been the topic of many physicostructural investigations both in solution and in the solid-state. Despite a large number of studies, the fundamental correlations between compositions, local symmetry, electronic structures of [{Ta6Bri12}La6]m+/n- cluster units (L = Br or H2O, in solution and in the solid-state), redox states, and vibrational and absorption properties are still not well established. Using K4[{Ta6Bri12}Bra6] as a starting precursor (i: inner and a: apical), we have investigated the behavior of the [{Ta6Bri12}Bra6]4- cluster unit in terms of oxidation properties and chemical modifications both in solution (water and organic solvent) and after recrystallization. A wide range of experimental techniques in combination with quantum chemical simulations afford new data that allow the puzzling behavior of the cluster units in response to changes in their environment to be revealed. Apical ligands undergo changes like modifications of interatomic distances to complete substitutions in solution that modify noticeably the cluster physical properties. Changes in the oxidation state of the cluster units also occur, which modify significantly their physical properties, including optical properties, which can thus be used as fingerprints. A subtle balance exists between the number of substituted apical ligands and the cluster oxidation state. This study provides new information about the exact nature of the species formed during the transition from the solid-state to solutions and vice versa. This shows new perspectives on optimization protocols for the design of Ta6 cluster-based materials.

Mass spectrometry is widely used in molecular science, and is now emerging as a characterization technique for ultra-small nanoparticles.

A pump–probe approach was designed to determine the internal proton transfer (PT) rate in a series of poly-peptide radical cations containing both histidine and tryptophan. The proton transfer is driven by the gas-phase basicity difference between residues. The fragmentation scheme indicates that the gas-phase basicity of histidine is lower than that of radical tryptophan so that histidine is always pulling the proton away from tryptophan. However, the proton transfer requires the two basic sites to be in close proximity, which is rate limited by the peptide conformational dynamics. PT rate measurements were used to probe and explore the peptide conformational dynamics in several poly-glycines/prolines/alanines. For small and unstructured peptides, the PT rate decreases with the size, as expected from a statistical point of view in a flat conformational space. Conversely, if structured conformations are accessible, the structural flexibility of the peptide is decreased. This slows down the occurrence of conformations favorable to proton transfer. A dramatic decrease in the PT rates was observed for peptides HAnW, when n changes from 5 to 6. This is attributed to the onset of a stable helix for n = 6. No such discontinuity is observed for poly-glycines or poly-prolines. In HAnW, the gas-phase basicity and helix propensity compete for the position of the charge. Interestingly, in this competition between PT and helix formation in HA6W, the energy gain associated with helix formation is large enough to slow down the PT beyond experimental time but does not ultimately prevail over the proton preference for histidine.

Liquid chromatography with UV detection and ion mobility-mass spectrometry are evaluated to study the E → Z thermal back relaxation kinetics of azobenzenes.

Atomically precise Au25(MBA)18 nanoclusters were investigated by mass spectrometry and ion mobility spectrometry. We show that clusters sharing the same chemical composition and bearing the same net charge may display different structures and different charge repartition patterns, namely, the number of charges corresponding to deprotonation of the ligand moieties through carboxyl groups is not the same for all detected species. Part of the observed heterogeneity is a consequence of spontaneous electron loss occurring in the gas phase, which modifies the net charge of the clusters while maintaining the initial (de)protonation state.