Human plasma IgG1 repertoires are simple, unique, and dynamic.

Although humans can produce billions of IgG1 variants through recombination and hypermutation, the diversity of IgG1 clones circulating in human blood plasma has largely eluded direct characterization. Here, we combined several mass-spectrometry-based approaches to reveal that the circulating IgG1 repertoire in human plasma is dominated by a limited number of clones in healthy donors and septic patients. We observe that each individual donor exhibits a unique serological IgG1 repertoire, which remains stable over time but can adapt rapidly to changes in physiology. We introduce an integrative protein- and peptide-centric approach to obtain and validate a full sequence of an individual plasma IgG1 clone de novo. This IgG1 clone emerged at the onset of a septic episode and exhibited a high mutation rate (13%) compared with the closest matching germline DNA sequence, highlighting the importance of de novo sequencing at the protein level. A record of this paper's transparent peer review process is included in the supplemental information.

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry.

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

Gas-Phase Photoluminescence and Photodissociation of Silver-Capped Hexagold Clusters.

We report on the radiative and nonradiative deactivation pathways of selected charge states of the stoichiometric hexagold phosphine-stabilized ionic clusters, [(C)(AuDppy)6Ag2·(BF4) x](4- x)+ with x = 2 and 3 (Dppy = diphenylphosphino-2-pyridine), combining gas-phase photoluminescence and photodissociation with quantum chemical computations. These clusters possess an identical isostructural core made of a hyper-coordinated carbon at their center octahedrally surrounded by six gold ions, and two silver ions at their apexes. Their luminescence and fragmentation behavior upon photoexcitation was investigated under mass and charge control in an ion trap. The experimental and computational results shed light on the electronic states involved in the optical transitions as well as on their core, ligand, or charge transfer character. Gas-phase results are discussed in relation with condensed phase measurements, as well as previous observations in solution and on metal-organic frameworks. The monocationic species ( x = 3) is found to be less stable than the dicationic one ( x = 2). In the luminescence spectrum of the monocationic species, a shoulder at short wavelength can be observed and is assigned to fragment emission. This fragment formation appears to be favored for the monocation by the existence of a low lying singlet state energetically overlapping with the triplet state manifold, which is populated quickly after photoexcitation.

Vibronic Coupling Analysis of the Ligand-Centered Phosphorescence of Gas-Phase Gd(III) and Lu(III) 9-Oxophenalen-1-one Complexes.

The gas-phase laser-induced photoluminescence of cationic mononuclear gadolinium and lutetium complexes involving two 9-oxophenalen-1-one ligands is reported. Performing measurements at a temperature of 83 K enables us to resolve vibronic transitions. Via comparison to Franck-Condon computations, the main vibrational contributions to the ligand-centered phosphorescence are determined to involve rocking, wagging, and stretching of the 9-oxophenalen-1-one-lanthanoid coordination in the low-energy range, intraligand bending, and stretching in the medium- to high-energy range, rocking of the carbonyl and methine groups, and C-H stretching beyond. Whereas Franck-Condon calculations based on density-functional harmonic frequency computations reproduce the main features of the vibrationally resolved emission spectra, the absolute transition energies as determined by density functional theory are off by several thousand wavenumbers. This discrepancy is found to remain at higher computational levels. The relative energy of the Gd(III) and Lu(III) emission bands is only reproduced at the coupled-cluster singles and doubles level and beyond.

Correlation of the structural information obtained for europium-chelate ensembles from gas-phase photoluminescence and ion-mobility spectroscopy with density-functional computations and ligand-field theory.

We report a combined investigation of europium(iii)9-oxo-phenalen-1-one (PLN) coordination complexes, [Eu(PLN)4AE]+ with AE = Mg, Ca, and Sr, using gas-phase photoluminescence, trapped ion-mobility spectrometry and density-functional computations. In order to sort out the structural impact of the alkali earth dications on the photoluminescence spectra, the experimental data are compared to the predicted ligand-field splittings as well as to the collision cross-sections for different isomers of [Eu(PLN)4AE]+. The best fitting interpretation is that one isomer family predominantly contributes to the recorded luminescence. The present work demonstrates the complexity of the coordination patterns of multicenter lanthanoid chelates involved in dynamical equilibria and the pertinence of using isolation techniques to elucidate their photophysical properties.

Divergent Coordination Chemistry: Parallel Synthesis of [2×2] Iron(II) Grid-Complex Tauto-Conformers.

The coordination of iron(II) ions by a homoditopic ligand L with two tridentate chelates leads to the tautomerism-driven emergence of complexity, with isomeric tetramers and trimers as the coordination products. The structures of the two dominant [Fe(II) 4 L4 ](8+) complexes were determined by X-ray diffraction, and the distinctness of the products was confirmed by ion-mobility mass spectrometry. Moreover, these two isomers display contrasting magnetic properties (Fe(II) spin crossover vs. a blocked Fe(II) high-spin state). These results demonstrate how the coordination of a metal ion to a ligand that can undergo tautomerization can increase, at a higher hierarchical level, complexity, here expressed by the formation of isomeric molecular assemblies with distinct physical properties. Such results are of importance for improving our understanding of the emergence of complexity in chemistry and biology.

Gas-Phase Photoluminescence Characterization of Stoichiometrically Pure Nonanuclear Lanthanoid Hydroxo Complexes Comprising Europium or Gadolinium.

Gas-phase photoluminescence measurements involving mass-spectrometric techniques enable determination of the properties of selected molecular systems with knowledge of their exact composition and unaffected by matrix effects such as solvent interactions or crystal packing. The resulting reduced complexity facilitates a comparison with theory. Herein, we provide a detailed report of the intrinsic luminescence properties of nonanuclear europium(III) and gadolinium(III) 9-hydroxyphenalen-1-one (HPLN) hydroxo complexes. Luminescence spectra of [Eu9(PLN)16(OH)10](+) ions reveal an europium-centered emission dominated by a 4-fold split Eu(III) hypersensitive transition, while photoluminescence lifetime measurements for both complexes support an efficient europium sensitization via a PLN-centered triplet-state manifold. The combination of gas-phase measurements with density functional theory computations and ligand-field theory is used to discuss the antiprismatic core structure of the complexes and to shed light on the energy-transfer mechanism. This methodology is also employed to fit a new set of parameters, which improves the accuracy of ligand-field computations of Eu(III) electronic transitions for gas-phase species.

Photoluminescence Spectroscopy of Mass-Selected Electrosprayed Ions Embedded in Cryogenic Rare-Gas Matrixes.

An apparatus is presented which combines nanoelectrospray ionization for isolation of large molecular ions from solution, mass-to-charge ratio selection in gas-phase, low-energy-ion-beam deposition into a (co-condensed) inert gas matrix and UV laser-induced visible-region photoluminescence (PL) of the matrix isolated ions. Performance is tested by depositing three different types of lanthanoid diketonate cations including also a dissociation product species not directly accessible by chemical synthesis. For these strongly photoluminescent ions, accumulation of some femto- to picomoles in a neon matrix (over a time scale of tens of minutes to several hours) is sufficient to obtain well-resolved dispersed emission spectra. We have ruled out contributions to these spectra due to charge neutralization or fragmentation during deposition by also acquiring photoluminescence spectra of the same ionic species in the gas phase.

Ion mobility spectrometry, infrared dissociation spectroscopy, and ab initio computations toward structural characterization of the deprotonated leucine-enkephalin peptide anion in the gas phase.

Although the sequencing of protonated proteins and peptides with tandem mass spectrometry has blossomed into a powerful means of characterizing the proteome, much less effort has been directed at their deprotonated analogues, which can offer complementary sequence information. We present a unified approach to characterize the structure and intermolecular interactions present in the gas-phase pentapeptide leucine-enkephalin anion by several vibrational spectroscopy schemes as well as by ion-mobility spectrometry, all of which are analyzed with the help of quantum-chemical computations. The picture emerging from this study is that deprotonation takes place at the C terminus. In this configuration, the excess charge is stabilized by strong intramolecular hydrogen bonds to two backbone amide groups and thus provides a detailed picture of a potentially common charge accommodation motif in peptide anions.

Effect of Proton Substitution by Alkali Ions on the Fluorescence Emission of Rhodamine B Cations in the Gas Phase.

The photophysics of chromophores is strongly influenced by their environment. Solvation, charge state, and adduct formation significantly affect ground and excited state energetics and dynamics. The present study reports on fluorescence emission of rhodamine B cations (RhBH+) and derivatives in the gas phase. Substitution of the acidic proton of RhBH+ by alkali metal cations, M+, ranging from lithium to cesium leads to significant and systematic blue shifts of the emission. The gas-phase structures and singlet transition energies of RhBH+ and RhBM+, M = Li, Na, K, Rb, and Cs, were investigated using Hartree-Fock theory, density functional methods, second-order Møller-Plesset perturbation theory, and the second-order approximate coupled-cluster model CC2. Comparison of experimental and theoretical results highlights the need for improved quantum chemical methods, while the hypsochromic shift observed upon substitution appears best explained by the Stark effect due to the inhomogeneous electric field generated by the alkali ions.

Characterization of Nonanuclear Europium and Gadolinium Complexes by Gas-Phase Luminescence Spectroscopy.

Gas-phase measurements using mass-spectrometric techniques allow determination of the luminescence properties of selected molecular systems with knowledge of their exact composition. Furthermore, isolated luminophores are unaffected by matrix effects like solvent interactions or crystal packing. As a result, the system complexity is reduced relative to the condensed phase and a direct comparison with theory is facilitated. Herein, we report the intrinsic luminescence properties of nonanuclear europium(III) and gadolinium(III) 9-hydroxyphenalen-1-one (HPLN)-hydroxo complexes. Luminescence spectra of [Eu9(PLN)16(OH)10](+) ions reveal an europium-centered emission dominated by a 4-fold split Eu(III) hypersensitive transition. The corresponding Gd(III) complex, [Gd9(PLN)16(OH)10](+), shows a broad emission from a ligand based triplet state with an onset of about 1000 wavenumbers in excess of the europium emission. As supported by photoluminescence lifetime measurements for both complexes, we deduce an efficient europium sensitization via PLN-based triplet states. The luminescence spectra of the complexes are discussed in terms of a square antiprismatic europium/gadolinium core structure as suggested by density functional computations.

Substitutional photoluminescence modulation in adducts of a europium chelate with a range of alkali metal cations: a gas-phase study.

We present gas-phase dispersed photoluminescence spectra of europium(III) 9-hydroxyphenalen-1-one (HPLN) complexes forming adducts with alkali metal ions ([Eu(PLN)3M](+) with M = Li, Na, K, Rb, and Cs) confined in a quadrupole ion trap for study. The mass selected alkali metal cation adducts display a split hypersensitive (5)D0 → (7)F2 Eu(3+) emission band. One of the two emission components shows a linear dependence on the radius of the alkali metal cation whereas the other component displays a quadratic dependence thereon. In addition, the relative intensities of both components invert in the same order. The experimental results are interpreted with the support of density functional calculations and Judd-Ofelt theory, yielding also structural information on the isolated [Eu(PLN)3M](+) chromophores.

Intrinsic fluorescence properties of rhodamine cations in gas-phase: triplet lifetimes and dispersed fluorescence spectra.

We have investigated the gas phase triplet state lifetimes and dispersed fluorescence spectra of several types of rhodamine cations confined in a quadrupole ion trap and thermalized to 85 K. The measured triplet lifetimes of rhodamine cations Rh6G(+), Rh575(+), RhB(+), and Rh101(+) are found to be on the order of seconds, several orders of magnitude longer than those typically observed for the same dyes in optical condensed phase measurements. In addition dispersed fluorescence emission spectra in the gas phase at 85 K have been measured. The experimental gas phase results as well as solution measurements are compared to density functional calculations and the previous literature. Possible explanations for the discrepancy of gas and solution phase triplet lifetimes are discussed.

Gas phase fullerene anions hydrogenation by methanol followed by IRMPA dehydrogenation.

The characterization in the gas phase of the mechanisms responsible for hydride formation can contribute to the development of new materials for hydrogen storage. The present work provides evidence of a hydrogenation-dehydrogenation catalytic cycle for C(60)(*-) anions in the gas phase using methanol vapor at room temperature as hydrogen donor. The involvement of methanol in the reaction is confirmed by experiments using CD(3)OD and CD(3)OH. C(60) hydride anions with up to 11 hydrogen atoms are identified via elemental composition analysis using FT-ICR mass spectrometry. For the longer reaction times, partial conversion of the C(60) hydride ions into oxygen containing ion products occurs. Dehydrogenation using infrared multiphoton activation with a CO(2) laser restores the C(60)(*-) anions.