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Gene therapies based on adeno-associated viruses are an emerging area with high potential to improve human health. Current quality control techniques to assess contaminates and byproducts from the adeno-associated virus (AAV) production pipelines are lacking in robustness and throughput. To address these limitations, we coupled an automated microfluidic device called SampleStream with Orbitrap-based charge detection mass spectrometry (SS-CDMS). We demonstrate that the SS-CDMS workflow performs AAV analysis in under 15 min per sample in a completely autonomous manner. The SS-CDMS workflow enables rapid assessment of key quality control attributes (CQAs), such as of molecular weight and content ratio of AAV formulations with a small sample requirement (<2 × 10 9 capsids) without being limited by sample concentration. Additionally, this work shows the potential for the SS-CDMS workflow to be implemented at various stages of the production pipeline through effective sample clean up from more complex AAV matrices such as cell culture media.

Individual ion mass spectrometry (I 2 MS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, I 2 MS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. I 2 MS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing I 2 MS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns., Competing Interests: Competing interests: The authors declare the following competing financial interest(s): P.F.Y., V.Z., K.S. and M.W.S. are employees of Thermo Fisher Scientific, a provider of MS systems. R.T.F., K.R.D., J.B.G. and N.L.K. are involved in commercialization of MS informatics software including STORIboard. N.L.K. is a paid consultant for Thermo Fisher Scientific., (© 2025. Springer Nature Limited.)

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I 2 MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I 2 MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I 2 MS spectra that contained a positive COVID-19 antibody response and 21 I 2 MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I 2 MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.

We introduce single cell Proteoform imaging Mass Spectrometry ( scPiMS ), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.

The quality of data in charge detection mass spectrometry depends on accurate determination of ion charge. While the method of selective temporal overview of resonant ions (STORI) has proven to be highly enabling for determining the charge of ions that survive for variable amounts of time, it assumes that the ion frequency exactly matches the frequency being used in the calculation. Any mismatches result in low charge estimates. To address this, the misSTORI method was developed to correct these discrepancies. This can significantly reduce the charge measurement errors for samples with unstable masses. As an example, the misSTORI approach can eliminate a 5.7% charge determination error for a VP3-only AAV capsid that shifts 25 ppm in mass.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I 2 MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I 2 MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I 2 MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N -glycans followed by I 2 MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I 2 MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I 2 MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I 2 MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.

Collision cross section (CCS) measurements determined by ion mobility spectrometry (IMS) provide useful information about gas-phase protein structure that is complementary to mass analysis. Methods for determining CCS without a dedicated IMS system have been developed for Fourier transform mass spectrometry (FT-MS) platforms by measuring the signal decay during detection. Individual ion mass spectrometry (I 2 MS) provides charge detection and measures ion lifetimes across the length of an FT-MS detection event. By tracking lifetimes for entire ion populations, we demonstrate simultaneous determination of charge, mass, and CCS for proteins and complexes ranging from ∼8 to ∼232 kDa.

The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS 2 ) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS 2 identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer., (© 2023. Springer Nature Limited.)

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with the mass-to-charge ratio ( m / z ). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.

Beyond the now well-known strong catalyst-support interactions reported for ceria-supported platinum catalysts, intermetallic Ce-Pt compounds exhibit fascinating properties such as heavy fermion behavior and magnetic instability. Small heterometallic Ce-Pt clusters, which can provide insights into the local features that govern bulk phenomena, have been less explored. Herein, the anion photoelectron spectra of three small mixed Ce-Pt clusters, Ce 2 OPt - , Ce 2 Pt - , and Ce 3 Pt - , are presented and interpreted with supporting density functional theory calculations. The calculations, which are readily reconciled with the experimental spectra, suggest the presence of numerous close-lying spin states, including states in which the Ce 4 f electrons are ferromagnetically coupled or antiferromagnetically coupled. The Pt center is consistently in a nominal -2 charge state in all cluster neutrals and anions, giving the Ce-Pt bond ionic character. Ce-Pt bonds are stronger than Ce-Ce bonds, and the O atom in Ce 2 OPt - coordinates only with the Ce centers. The energy of the singly occupied Ce-local 4 f orbitals relative to the Pt-local orbitals changes with cluster composition. Discussion of the results includes potential implications for Ce-rich intermetallic materials.

Charge detection mass spectrometry (CDMS) provides mass domain spectra of large and highly heterogeneous analytes. Over the past few years, we have multiplexed CDMS on Orbitrap instruments, an approach termed Individual Ion Mass Spectrometry (I 2 MS). Until now, I 2 MS required manual adjustment of injection times to collect spectra in the individual ion regime. To increase sample adaptability, enable online separations, and reduce the barrier for entry, we report an automated method for adjusting ion injection times in I 2 MS for image current detectors like the Orbitrap. Automatic Ion Control (AIC) utilizes the density of signals in the m / z domain to adjust an ensemble of ions down to the individual ion regime in real-time. The AIC technique was applied to both denatured and native proteins yielding high quality data without human intervention directly in the mass domain.

SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron variants more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than their predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that (1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and (2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-μm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high m / z by >2.5-fold and >1.6-fold, respectively.

An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I 2 MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.

Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (I 2 MS) and spectral output directly into the mass domain. Here I 2 MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I 2 MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I 2 MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.

New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of so-called "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher m / z with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions. Herein, these differences are explored both theoretically and experimentally to understand the role that charge state and isotopic distributions have on signal-to-noise (S/N) as a function of complex molecular weight and how the reduced collisional cross sections of proteins electrosprayed under native solution conditions can lead to improved data quality in image current mass analyzers, such as Orbitrap and FT-ICR. Quantifying ion signal differences under native and denatured conditions revealed enhanced S/N and a more gradual decay in S/N with increasing mass under native conditions. Charge state and isotopic S/N models, supported by experimental results, indicate that analysis of proteins under native conditions at 100 kDa will be 17 times more sensitive than analysis under denatured conditions at the same mass. Higher masses produce even larger sensitivity gains. Furthermore, reduced cross sections under native conditions lead to lower levels of ion decay within an Orbitrap scan event over long transient acquisition times, enabling isotopic resolution of species with molecular weights well in excess of those typically resolved under denatured conditions.

Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.

Native mass spectrometry (nMS) is a technique growing at the interface of analytical chemistry, structural biology, and proteomics that enables the detection and partial characterization of non-covalent protein assemblies. Currently, the standardization and dissemination of nMS is hampered by technical challenges associated with instrument operation, benchmarking, and optimization over time. Here, we provide a standard operating procedure for acquiring high-quality native mass spectra of 30-300 kDa proteins using an Orbitrap mass spectrometer. By describing reproducible sample preparation, loading, ionization, and nMS analysis, we forward two proteoforms and three complexes as possible standards to advance training and longitudinal assessment of instrument performance. Spectral data for five standards can guide assessment of instrument parameters, data production, and data analysis. By introducing this set of standards and protocols, we aim to help normalize native mass spectrometry practices across labs and provide benchmarks for reproducibility and high-quality data production in the years ahead. Graphical abstract.

It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/ z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.

Lanthanide (Ln) oxide clusters have complex electronic structures arising from the partially occupied Ln 4f subshell. New anion photoelectron (PE) spectra of Sm x Ce 3-x O y - (x = 0-3; y = 2-4) along with supporting results of density functional theory (DFT) calculations suggest interesting x and y-dependent Sm 4f subshell occupancy with implications for Sm-doped ionic conductivity of ceria, as well as the overall electronic structure of the heterometallic oxides. Specifically, the Sm centers in the heterometallic species have higher 4f subshell occupancy than the homonuclear Sm 3 O y - /Sm 3 O y clusters. The higher 4f subshell occupancy both weakens Sm-O bonds and destabilizes the 4f subshell relative to the predominantly O 2p bonding orbitals in the clusters. Parallels between the electronic structures of these small cluster systems with bulk oxides are explored. In addition, unusual changes in the excited state transition intensities, similar to those observed previously in the PE spectra of Sm 2 O - [J. O. Kafader et al., J. Chem. Phys. 146, 194310 (2017)], are also observed in the relative intensities of electronic transitions to excited neutral state bands in the PE spectra of Sm 2 O 2 - [J. O. Kafader et al., J. Chem. Phys. 146, 194310 (2017)], are also observed in the relative intensities of electronic transitions to excited neutral state bands in the PE spectra of Sm x Ce 3-x O y - (x = 1-3; y = 2, 4). The new spectra suggest that the effect is enhanced with lower oxidation states and with an increasing number of Sm atoms, implying that the prevalence of electrons in the diffuse Sm 6s-based molecular orbitals and a more populated 4f subshell both contribute to this phenomenon. Finally, this work identifies challenges associated with affordable DFT calculations in treating the complex electronic structures exhibited by these systems, including the need for a more explicit treatment of strong coupling between the neutral and PE.

To probe the mechanism of sacrificial reagents in catalytic processes, product distributions from Mo x O y - clusters reacting individually with C 2 H 4 and H 2 O are compared with those from reactions with a C 2 H 4 + H 2 O mixture, with the thermodynamics explored computationally. These molecules were chosen to model production of H 2 from H 2 O via H 2 O + C 2 H 4 → H 2 + CH 3 CHO, mediated by Mo x O y - clusters. H 2 O is known to sequentially oxidize Mo x O y - suboxide clusters while producing H 2 , resulting in less reactive clusters. Mo x O y - (y ∼ x) clusters undergo chemi-fragmentation reactions with C 2 H 4 , with Mo x O y C 2 H z - complexes forming as the cluster oxidation state increases. Unique species observed in reactions with the C 2 H 4 + H 2 O mixture, Mo 2 O 5 C 2 H 2 - and MoO 3 C 2 H 4 - , suggest that the internal energy gained in new Mo-O bond formation from oxidation by H 2 O opens additional reaction channels. C 2 H 3 O - is observed uniquely in reactions with the C 2 H 4 + H 2 O mixture, giving indirect evidence of CH 3 CHO formation via the cluster mediated H 2 O + C 2 H 4 → H 2 + CH 3 CHO reaction; C 2 H 3 O - can form via dissociative electron attachment to CH 3 CHO. Calculations support mechanisms that invoke participation of two ethylene molecules on thermodynamically favorable pathways leading to experimentally observed products.

The anion photoelectron (PE) spectra of a range of small mono-cerium molecular species, along with the Ce 2 O 4 - and Ce 3 O 6 - stoichiometric clusters, are presented and analyzed with the support of density functional theory calculations. A common attribute of all of the neutral species is that the Ce centers in both the molecules and clusters are in the +4 oxidation state. In bulk ceria (CeO 2 ), an unoccupied, narrow 4f band lies between the conventional valence (predominantly O 2p) and conduction (Ce 5d) bands. Within the CeO 2 - , CeO 3 H 2 - , and Ce(OH) 4 - series, the PE spectra and computational results suggest that the Ce 6s-based molecular orbital is the singly occupied HOMO in CeO 2 - but becomes destabilized as the Ce 4f-local orbital becomes stabilized with increasing coordination. CeO 3 - , a hyperoxide, undergoes photodissociation with 3.49 eV photon energy to form the stoichiometric neutral CeO 2 and O - . In the CeO 2 - , Ce 2 O 4 - ,and Ce 3 O 6 - stoichiometric cluster series, the 6s destabilization with 4f stabilization is associated with increasing cluster size, suggesting that a bulk-like band structure may be realized with fairly small cluster sizes. The destabilization of the 6s-based molecular orbitals can be rationalized by their diffuse size relative to Ce-O bond lengths in a crystal structure, suggesting that 6s bands in the bulk may be relegated to the surface.

The anion photoelectron (PE) spectra along with supporting results of density functional theory (DFT) calculations on SmO - , SmCeO y - , and Sm 2 O y - (y = 1, 2) are reported and compared to previous results on CeO - (y = 1, 2) [J. O. Kafader et al., J. Chem. Phys. 145, 154306 (2016)]. Similar to the results on Ce 2 O y - (y = 1, 2) [J. O. Kafader et al., J. Chem. Phys. 145, 154306 (2016)]. Similar to the results on Ce x O y - clusters, the PE spectra of SmO - , SmCeO y - , and Sm 2 O y - (y = 1, 2) all exhibit electronic transitions to the neutral ground state at approximately 1 eV e - BE. The Sm centers in SmO and Sm 2 O 2 neutrals can be described with the 4f 5 6s superconfiguration, which is analogous to CeO and Ce 2 O 2 neutrals in which the Ce centers can be described with the 4f 6s superconfiguration (Z Ce = Z Sm - 4). The Sm center in CeSmO 2 , in contrast, has a 4f 6 occupancy, while the Ce center maintains the 4f 6s superconfiguration. The less oxidized Sm centers in both Sm 2 O and SmCeO have 4f 6 6s occupancies. The 4f 6 subshell occupancy results in relatively weak Sm-O bond strengths. If this extra 4f occupancy also occurs in bulk Sm-doped ceria, it may play a role in the enhanced O 2- ionic conductivity in Sm-doped ceria. Based on the results of DFT calculations, the heteronuclear Ce-Sm oxides have molecular orbitals that are distinctly localized Sm 4f, Sm 6s, Ce 4f, and Ce 6s orbitals. The relative intensity of two electronic bands in the PE spectrum of Sm 2 O - exhibits an unusual photon energy-dependence, and the PE spectrum of Sm 2 O 2 - exhibits a photon energy-dependent continuum signal between two electronic transitions. Several explanations, including the high magnetic moment of these suboxide species and the presence of low-lying quasi-bound anion states, are considered.

The anion photoelectron (PE) spectra of Ce 2 O y - (y = 1, 2), Ce 3 O y - (y = 0-4), Ce 4 O y - (y = 0-2), and Ce 5 O y - (y = 1, 2) are reported and analyzed with supporting results from density functional theory calculations. The PE spectra all exhibit an intense electronic transition to the neutral ground state, all falling in the range of 0.7 to 1.1 eV electron binding energy, with polarization dependence consistent with detachment from diffuse Ce 6s-based molecular orbitals. There is no monotonic increase in electron affinity with increasing oxidation. A qualitative picture of how electronic structure evolves with an oxidation state emerges from comparison between the spectra and the computational results. The electronic structure of the smallest metallic cluster observed in this study, Ce 3 , is similar to the bulk structure in terms of atomic orbital occupancy (4f 5d 2 6s). Initial cerium cluster oxidation involves largely ionic bond formation via Ce 5d and O 2p orbital overlap (i.e., larger O 2p contribution), with Ce-O-Ce bridge bonding favored over Ce=O terminal bond formation. With subsequent oxidation, the Ce 5d-based molecular orbitals are depleted of electrons, with the highest occupied orbitals described as diffuse Ce 6s based molecular orbitals. In the y ≤ (x + 1) range of oxidation states, each Ce center has a singly occupied non-bonding 4f orbital. The PE spectrum of Ce 3 O 4 - is unique in that it exhibits a single nearly vertical transition. The highly symmetric structure predicted computationally is the same structure determined from Ce 3 O 4 + IR predissociation spectra [A. M. Burow et al., Phys. Chem. Chem. Phys. 13, 19393 (2011)], indicating that this structure is stable in -1, 0, and +1 charge states. Spectra of clusters with x ≥ 3 exhibit considerable continuum signal above the ground state transition; the intensity of the continuum signal decreases with increasing oxidation. This feature is likely the result of numerous quasi-bound anion states or two-electron transitions possible in molecules with abundant nearly degenerate partially occupied orbitals.

The electronic structures of several small Ce-Pt oxide complexes were explored using a combination of anion photoelectron (PE) spectroscopy and density functional theory calculations. Pt and Pt2 both accept electron density from CeO diatomic molecules, in which the cerium atom is in a lower-than-bulk oxidation state (+2 versus bulk +4). Neutral [CeO]Pt and [CeO]Pt2 complexes are therefore ionic, with electronic structures described qualitatively as [CeO(+2)]Pt(-2) and [CeO(+)]Pt2 (-), respectively. The associated anions are described qualitatively as [CeO(+)]Pt(-2) and [CeO(+)]Pt2 (-2), respectively. In both neutrals and anions, the most stable molecular structures determined by calculations feature a distinct CeO moiety, with the positively charged Ce center pointing toward the electron rich Pt or Pt2 moiety. Spectral simulations based on calculated spectroscopic parameters are in fair agreement with the spectra, validating the computationally determined structures. In contrast, when Pt is coupled with CeO2, which has no Ce-localized electrons that can readily be donated to Pt, the anion is described as [CeO2]Pt(-). The molecular structure predicted computationally suggests that it is governed by charge-dipole interactions. The neutral [CeO2]Pt complex lacks charge-dipole stabilizing interactions, and is predicted to be structurally very different from the anion, featuring a single Pt-O-Ce bridge bond. The PE spectra of several of the complexes exhibit evidence of photodissociation with Pt(-) daughter ion formation. The electronic structures of these complexes are related to local interactions in Pt-ceria catalyst-support systems.

Results of a systematic comparison of the MoxOy (-) + H2O and WxOy (-) + H2O reaction rate coefficients are reported and compared to previous experimental and computational studies on these reactions. WxOy (-) clusters undergo more direct oxidation by water to yield WxOy+1 (-) + H2, while for MoxOy (-) clusters, production of MoxOyH2 (-) (trapped intermediates in the oxidation reaction) is comparatively more prevalent. However, MoxOy (-) clusters generally have higher rate coefficients than analogous WxOy (-) clusters if MoxOy+1H2 (-) formation is included. Results of calculations on the M2Oy (-) + H2O (M = Mo, W; y = 4, 5) reaction entrance channel are reported. They include charge-dipole complexes formed from long-range interactions, and the requisite conversion to a Lewis acid-base complex that leads to MxOy+1H2 (-) formation. The results predict that the Lewis acid-base complex is more strongly bound for MoxOy (-) clusters than for WxOy (-) clusters. The calculated free energies along this portion of the reaction path are also consistent with the modest anti-Arrhenius temperature dependence measured for most MoxOy (-) + H2O reactions, and the WxOy (-) + H2O reaction rate coefficients generally being constant over the temperature range sampled in this study. For clusters that exhibit evidence of both water addition and oxidation reactions, increasing the temperature increases the branching ratio toward oxidation for both species. A more direct reaction path to H2 production may therefore become accessible at modest temperatures for certain cluster stoichiometries and structures.

The photoelectron (PE) spectrum of PrO(-) exhibits a short 835 ± 20 cm(-1) vibrational progression of doublets (210 ± 30 cm(-1) splitting) assigned to transitions from the 4f(2) [(3)H4] σ6s (2) Ω = 4 anion ground state to the 4f(2) [(3)H4] σ6s Ω = 3.5 and 4.5 neutral states. This assignment is analogous to that of the recently reported PE spectrum of CeO(-), though the 82 cm(-1) splitting between the 4f [(2)F2.5] σ6s Ω = 2 and Ω = 3 CeO neutral states could not be resolved [Ray et al., J. Chem. Phys. 142, 064305 (2015)]. The origin of the transition to the Ω = 3.5 neutral ground state is 0.96 ± 0.01 eV, which is the adiabatic electron affinity of PrO. Density functional theory calculations on the anion and neutral molecules support the assignment. The appearance of multiple, irregularly spaced and low-intensity features observed ca. 1 eV above the ground state cannot be reconciled with low-lying electronic states of PrO that are accessible via one-electron detachment. However, neutral states correlated with the 4f(2) [(3)H4] 5d superconfiguration are predicted to be approximately 1 eV above the 4f(2) [(3)H4] σ6s Ω = 3.5 neutral ground state, leading to the assignment of these features to shake-up transitions to the excited neutral states. Based on tentative hot band transition assignments, the term energy of the previously unobserved 4f(2) [(3)H4] σ6s Ω = 2.5 neutral state is determined to be 1840 ± 110 cm(-1).

The anion photoelectron (PE) spectra of EuH(-) and the PE spectrum of overlapping EuOH(-) and EuO(-) anions are presented and analyzed with supporting results from density functional theory calculations on the various anions and neutrals. Results point to ionically bound, high-spin species. EuH and EuOH anions and neutrals exhibit analogous electronic structures: Transitions from (8)Σ(-) anion ground states arising from the 4f(7)σ(6s)(2) superconfiguration to the close-lying neutral (9)Σ(-) and (7)Σ(-) states arising from the 4f(7)σ(6s) superconfiguration are observed spaced by an energy interval similar to the free Eu(+) [4f(7)6s] (9)S - (7)S splitting. The electron affinities (EAs) of EuH and EuOH are determined to be 0.771 ± 0.009 eV and 0.700 ± 0.011 eV, respectively. Analysis of spectroscopic features attributed to EuO(-) photodetachment is complicated by the likely presence of two energetically competitive electronic states of EuO(-) populating the ion beam. However, based on the calculated relative energies of the close-lying anion states arising from the 4f(7)σ(6s) and 4f(6)σ(6s)(2) configurations and the relative energies of the one-electron accessible 4f(7) and 4f(6)σ(6s) neutral states based on ligand-field theory [M. Dulick, E. Murad, and R. F. Barrow, J. Chem. Phys. 85, 385 (1986)], the remaining features are consistent with the 4f(6)σ(6s)(2) (7)Σ(-) and 4f(7)σ(6s) (7)Σ(-) anion states lying very close in energy (the former was calculated to be 0.15 eV lower in energy than the latter), though the true anion ground state and neutral EA could not be established unambiguously. Calculations on the various EuO anion and neutral states suggest 4f-orbital overlap with 2p orbitals in species with 4f(6) occupancy.

The photoelectron spectrum of CeO(-) exhibits what appears to be a single predominant electronic transition over an energy range in which numerous close-lying electronic states of CeO neutral are well known. The photoelectron spectrum of Ce(OH)2 (-), a molecule in which the Ce atom shares the same formal oxidation state as the Ce atom in CeO(-), also exhibits what appears to be a single transition. From the spectra, the adiabatic electron affinities of CeO and Ce(OH)2 are determined to be 0.936 ± 0.007 eV and 0.69 ± 0.03 eV, respectively. From the electron affinity of CeO, the CeO(-) bond dissociation energy was determined to be 7.7 eV, 0.5 eV lower than the neutral bond dissociation energy. The ground state orbital occupancies of both CeO(-) and Ce(OH)2 (-) are calculated to have 4f 6s(2) Ce(+) superconfigurations, with open-shell states having 4f5d6s superconfiguration predicted to be over 1 eV higher in energy. Low-intensity transitions observed at higher electron binding energies in the spectrum of CeO(-) are tentatively assigned to the (1)Σ(+) (Ω = 0) state of CeO with the Ce+26s2 superconfiguration.

Reactions between small cerium oxide cluster anions and deuterated water were monitored as a function of both water concentration and temperature in order to determine the temperature dependence of the rate constants. Sequential oxidation reactions of the Ce(x)O(y)⁻ (x = 2, 3) suboxide cluster anions were found to exhibit anti-Arrhenius behavior, with activation energies ranging from 0 to -18 kJ mol⁻¹. Direct oxidation of species up to y = x was observed, after which, -OD abstraction and D₂O addition reactions were observed. However, the stoichiometric Ce₂O₄⁻ and Ce₃O₆⁻ cluster anions also emerge in reactions between D₂O and the respective precursors, Ce₂O₃D⁻ and Ce₃O₅D₂⁻. Ce₂O₄⁻ and Ce₃O₆⁻ product intensities diminish relative to deuteroxide complex intensities with increasing temperature. The kinetics of these reactions are compared to the kinetics of the previously studied Mo(x)O(y)⁻ and W(x)O(y)⁻ reactions with water, and the possible implications for the reaction mechanisms are discussed.

Heat capacities have been measured as a function of temperature for size-selected gallium cluster cations with between 60 and 183 atoms. Almost all clusters studied show a single peak in the heat capacity that is attributed to a melting transition. The peaks can be fit by a two-state model incorporating only fully solid-like and fully liquid-like species, and hence no partially melted intermediates. The exceptions are Ga90(+), which does not show a peak, and Ga80(+) and Ga81(+), which show two peaks. For the clusters with two peaks, the lower temperature peak is attributed to a structural transition. The melting temperatures for clusters with less than 50 atoms have previously been shown to be hundreds of degrees above the bulk melting point. For clusters with more than 60 atoms the melting temperatures decrease, approaching the bulk value (303 K) at around 95 atoms, and then show several small upward excursions with increasing cluster size. A plot of the latent heat against the entropy change for melting reveals two groups of clusters: the latent heats and entropy changes for clusters with less than 94 atoms are distinct from those for clusters with more than 93 atoms. This observation suggests that a significant change in the nature of the bonding or the structure of the clusters occurs at 93-94 atoms. Even though the melting temperatures are close to the bulk value for the larger clusters studied here, the latent heats and entropies of melting are still far from the bulk values.