Anatomy of Protein Electrospray Ionization Mass Spectra by Superconducting Tunnel Junction Mass and Energy Spectrometry.

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-of-flight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear quadrupole ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of a fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is presumed to be a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

Feasibility of Laboratory-Based EXAFS Spectroscopy with Cryogenic Detectors.

Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy is a powerful technique that gives element-specific information about the structure of molecules. The development of a laboratory EXAFS spectrometer capable of measuring transmission spectra would be a significant advance as the technique is currently only available at synchrotron radiation lightsources. Here, we explore the potential of cryogenic detectors as the energy resolving component of a laboratory transmission EXAFS instrument. We examine the energy resolution, count-rate, and detector stability needed for good EXAFS spectra and compare these to the properties of cryogenic detectors and conventional X-ray optics. We find that superconducting tunnel junction (STJ) detectors are well-suited for this application.

L-edge sum rule analysis on 3d transition metal sites: from d10 to d0 and towards application to extremely dilute metallo-enzymes.

According to L-edge sum rules, the number of 3d vacancies at a transition metal site is directly proportional to the integrated intensity of the L-edge X-ray absorption spectrum (XAS) for the corresponding metal complex. In this study, the numbers of 3d holes are characterized quantitatively or semi-quantitatively for a series of manganese (Mn) and nickel (Ni) complexes, including the electron configurations 3d10→ 3d0. In addition, extremely dilute (<0.1% wt/wt) Ni enzymes were examined by two different approaches: (1) by using a high resolution superconducting tunnel junction X-ray detector to obtain XAS spectra with a very high signal-to-noise ratio, especially in the non-variant edge jump region; and (2) by adding an inert tracer to the sample that provides a prominent spectral feature to replace the weak edge jump for intensity normalization. In this publication, we present for the first time: (1) L-edge sum rule analysis for a series of Mn and Ni complexes that include electron configurations from an open shell 3d0 to a closed shell 3d10; (2) a systematic analysis on the uncertainties, especially on that from the edge jump, which was missing in all previous reports; (3) a clearly-resolved edge jump between pre-L3 and post-L2 regions from an extremely dilute sample; (4) an evaluation of an alternative normalization standard for L-edge sum rule analysis. XAS from two copper (Cu) proteins measured using a conventional semiconductor X-ray detector are also repeated as bridges between Ni complexes and dilute Ni enzymes. The differences between measuring 1% Cu enzymes and measuring <0.1% Ni enzymes are compared and discussed. This study extends L-edge sum rule analysis to virtually any 3d metal complex and any dilute biological samples that contain 3d metals.

Measurement of Small Molecular Dopant F4TCNQ and C60F36 Diffusion in Organic Bilayer Architectures.

The diffusion of molecules through and between organic layers is a serious stability concern in organic electronic devices. In this work, the temperature-dependent diffusion of molecular dopants through small molecule hole transport layers is observed. Specifically we investigate bilayer stacks of small molecules used for hole transport (MeO-TPD) and p-type dopants (F4TCNQ and C60F36) used in hole injection layers for organic light emitting diodes and hole collection electrodes for organic photovoltaics. With the use of absorbance spectroscopy, photoluminescence spectroscopy, neutron reflectometry, and near-edge X-ray absorption fine structure spectroscopy, we are able to obtain a comprehensive picture of the diffusion of fluorinated small molecules through MeO-TPD layers. F4TCNQ spontaneously diffuses into the MeO-TPD material even at room temperature, while C60F36, a much bulkier molecule, is shown to have a substantially higher morphological stability. This study highlights that the differences in size/geometry and thermal properties of small molecular dopants can have a significant impact on their diffusion in organic device architectures.

Soft X-ray absorption spectroscopy and resonant inelastic X-ray scattering spectroscopy below 100 eV: probing first-row transition-metal M-edges in chemical complexes.

X-ray absorption and scattering spectroscopies involving the 3d transition-metal K- and L-edges have a long history in studying inorganic and bioinorganic molecules. However, there have been very few studies using the M-edges, which are below 100 eV. Synchrotron-based X-ray sources can have higher energy resolution at M-edges. M-edge X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray scattering (RIXS) could therefore provide complementary information to K- and L-edge spectroscopies. In this study, M2,3-edge XAS on several Co, Ni and Cu complexes are measured and their spectral information, such as chemical shifts and covalency effects, are analyzed and discussed. In addition, M2,3-edge RIXS on NiO, NiF2 and two other covalent complexes have been performed and different d-d transition patterns have been observed. Although still preliminary, this work on 3d metal complexes demonstrates the potential to use M-edge XAS and RIXS on more complicated 3d metal complexes in the future. The potential for using high-sensitivity and high-resolution superconducting tunnel junction X-ray detectors below 100 eV is also illustrated and discussed.

Molybdenum X-ray absorption edges from 200 to 20,000eV: the benefits of soft X-ray spectroscopy for chemical speciation.

We have surveyed the chemical utility of the near-edge structure of molybdenum X-ray absorption edges from the hard X-ray K-edge at 20,000eV down to the soft X-ray M(4,5)-edges at approximately 230eV. We compared, for each edge, the spectra of two tetrahedral anions, MoO(4)(2-) and MoS(4)(2-). We used three criteria for assessing near-edge structure of each edge: (i) the ratio of the observed chemical shift between MoO(4)(2-) and MoS(4)(2-) and the linewidth, (ii) the chemical information from analysis of the near-edge structure and (iii) the ease of measurement using fluorescence detection. Not surprisingly, the K-edge was by far the easiest to measure, but it contained the least information. The L(2,3)-edges, although harder to measure, had benefits with regard to selection rules and chemical speciation in that they had both a greater chemical shift as well as detailed lineshapes which could be theoretically analyzed in terms of Mo ligand field, symmetry, and covalency. The soft X-ray M(2,3)-edges were perhaps the least useful, in that they were difficult to measure using fluorescence detection and had very similar information content to the corresponding L(2,3)-edges. Interestingly, the soft X-ray, low energy ( approximately 230eV) M(4,5)-edges had greatest potential chemical sensitivity and using our high-resolution superconducting tunnel junction (STJ) fluorescence detector they appear to be straightforward to measure. The spectra were amenable to analysis using both the TT-multiplet approach and FEFF. The results using FEFF indicate that the sharp near-edge peaks arise from 3d-->5p transitions, while the broad edge structure has predominately 3d-->4f character. A proper understanding of the dependence of these soft X-ray spectra on ligand field and site geometry is necessary before a complete assessment of the utility of the Mo M(4,5)-edges can be made. This work includes crystallographic characterization of sodium tetrathiomolybdate.

Cryogenic X-ray detectors for synchrotron science.

Cryogenic detectors operated at temperatures below 1 K offer an order of magnitude higher energy resolution than conventional semiconductor-based energy-dispersive detectors, and orders of magnitude higher efficiency that grating spectrometers. Initially developed for astrophysics applications, these detectors are increasingly used in synchrotron-based research, both for detector characterization and for high-resolution X-ray spectroscopy. This article reviews current cryogenic detector technologies and their performance with respect to their use in synchrotron science. It also discusses areas of research that can benefit from improved energy resolution detectors, and outlines desirable detector developments in the context of novel science they would enable.

Direct observation of nitrogen location in molecular beam epitaxy grown nitrogen-doped ZnO.

ZnO is a wide band gap, naturally n-type semiconductor with great promise for optoelectronic applications; the main obstacle yet to be overcome is p-type doping. Nitrogen, the most promising candidate currently being pursued as a dopant, has been predicted to preferentially incorporate into the ZnO lattice in the form of a N-2 molecule at an O site when a plasma source is used, leading to compensation rather than p-type doping. We demonstrate this to be incorrect by using N K-edge x-ray absorption spectra and comparing them with first-principles calculations showing that nitrogen, in fact, incorporates substitutionally at O sites where it is expected to act as an acceptor. We also detect the formation of molecular nitrogen upon annealing. These results suggest that effective p-type doping of ZnO with N may be possible only for low-temperature growth processes.

X-ray magnetic circular dichroism of Pseudomonas aeruginosa nickel(II) azurin.

We show that X-ray magnetic circular dichroism (XMCD) can be employed to probe the oxidation states and other electronic structural features of nickel active sites in proteins. As a calibration standard, we have measured XMCD and X-ray absorption (XAS) spectra for the nickel(II) derivative of Pseudomonas aeruginosa azurin (NiAz). Our analysis of these spectra confirms that the electronic ground state of NiAz is high-spin (S = 1); we also find that the L(3)-centroid energy is 853.1(1) eV, the branching ratio is 0.722(4), and the magnetic moment is 1.9(4) mu(B). Density functional theory (DFT) calculations on model NiAz structures establish that orbitals 3d(x2-y2) and 3d(z2) are the two valence holes in the high-spin Ni(II) ground state, and in accord with the experimentally determined orbital magnetic moment, the DFT results also demonstrate that both holes are highly delocalized, with 3d(x2-y2) having much greater ligand character.

Chemically distinct Ni sites in the A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni L-edge absorption and X-ray magnetic circular dichroism analyses.

The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.

Nearest-neighbor configuration in (GaIn)(NAs) probed by x-ray absorption spectroscopy.

Ga(1-x)In(x)N(y)As(1-y) is a promising material system for the fabrication of inexpensive "last-mile" optoelectronic components. However, details of its atomic arrangement and the relationship to observed optical properties is not fully known. Particularly, a blueshift of emission wavelength is observed after annealing. In this work, we use x-ray absorption fine structure to study the chemical environment around N atoms in the material before and after annealing. We find that as-grown molecular beam epitaxy material consists of a nearly random distribution of atoms, while postannealed material shows segregation of In toward N. Ab initio simulations show that this short-range ordering creates a more thermodynamically stable alloy and is responsible for blueshifting the emission.