Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high‐resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high‐performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI‐Orbitrap or the MALDI‐TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI‐Orbitrap mass spectrometer enabled differentiation between two possible RNA‐based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In‐source decay (ISD) analysis using a MALDI‐TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4‐dihydroxyacetophenone (2,4‐DHAP) as a matrix. The ESI‐Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on‐line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI‐TOF mass spectrometer, in combination with 2,4‐DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Full Scan MS in Comprehensive Qualitative and Quantitative Residue Analysis in Food and Feed Matrices: How Much Resolving Power is Required?

In LC full scan based MS screening methods correct mass assignment is essential. Parameters affecting the accuracy of mass assignment, i.e., analyte concentration, complexity of the matrix, and resolving power, were studied using typical examples from the field of residue and contaminant analysis in food and feed. The evaluation was carried out by analyzing samples of honey and animal feed, spiked with 151 pesticides, veterinary drugs, mycotoxins, and plant toxins at levels ranging from 10 to 250 ng/g. Analyses were performed using a single stage Orbitrap with resolving power settings varying from 10,000 to 100,000 (FWHM). For consistent and reliable mass assignment (<2 ppm) of analytes at low levels in complex matrices, a high resolving power (≥50,000) was found to be required. At lower resolving power settings, the error in the assignment of mass increased due to the coelution of analytes with interferences at the same nominal mass. This negatively affected selectivity and quantitative performance due to the inability to use the required narrow mass-extraction windows. In the case of the less complex honey matrix, a resolving power of 25,000 was generally sufficient to obtain a mass assignment error close to the typical instrument mass accuracy (≤2 ppm) down to low concentration levels of 10 ng/g.     

Spectral Accuracy and Sulfur Counting Capabilities of the LTQ-FT-ICR and the LTQ-Orbitrap XL for Small Molecule Analysis

Color Index Disperse Yellow 42 (DY42), a high-volume disperse dye for polyester, was used to compare the capabilities of the LTQ-Orbitrap XL and the LTQ-FT-ICR with respect to mass measurement accuracy (MMA), spectral accuracy, and sulfur counting. The results of this research will be used in the construction of a dye database for forensic purposes; the additional spectral information will increase the confidence in the identification of unknown dyes found in fibers at crime scenes. Initial LTQ-Orbitrap XL data showed MMAs greater than 3 ppm and poor spectral accuracy. Modification of several Orbitrap installation parameters (e.g., deflector voltage) resulted in a significant improvement of the data. The LTQ-FT-ICR and LTQ-Orbitrap XL (after installation parameters were modified) exhibited MMA ≤ 3 ppm, good spectral accuracy (χ² values for the isotopic distribution ≤ 2), and were correctly able to ascertain the number of sulfur atoms in the compound at all resolving powers investigated for AGC targets of 5.00 × 10⁵ and 1.00 × 10⁶.     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

MALDI produced ions inspected with a linear ion trap-orbitrap hybrid mass analyzer

A MALDI source is interfaced to a modified LTQ Orbitrap XL instrument. This work gives insight into the MALDI source design and shows results obtained with the MALDI source coupled to an accurate mass, high-resolution hybrid mass spectrometer. MALDI-produced ions and fragment ions thereof produced in the mass spectrometer may be analyzed and detected by the Orbitrap analyzer at a maximum mass resolution of 100,000 (FWHM) at m/z 400 with high mass accuracy. An accuracy of ≤2 ppm is achieved by internal mass calibration using lock mass functionality; using external mass calibration, an accuracy of ≤3 ppm is routinely obtained. External mass calibration of the hybrid mass spectrometer is performed using a standard calibration mixture of different peptides and matrix components. The instrumental capabilities are demonstrated for analytical methodologies such as Protein ID using Peptide Mass Fingerprint (PMF) and MS/MS analyses of small molecule samples. Stability of mass accuracy and signal-to-noise ratio for low samples loads (on plates) are demonstrated as well as the experimental dynamic range using α-cyano-4-hydroxy cinnamic acid (CHCA) matrix.     

Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses

The development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76 ppm and 1.62 ± 1.88 ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5 ppm for the Orbitrap and greater than 10 ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.

Accurate mass measurements and ultrahigh-resolution: evaluation of different mass spectrometers for daily routine analysis of small molecules in negative electrospray ionization mode

Six mass spectrometers based on different mass analyzer technologies, such as time-of-flight (TOF), hybrid quadrupole-TOF (Q-TOF), orbitrap, Fourier transform ion cyclotron resonance (FT-ICR), and triple quadrupole (QqQ), installed at independent laboratories have been tested during a single day of work for the analysis of small molecules in negative electrospray ionization (ESI) mode. The uncertainty in the mass measurements obtained from each mass spectrometer has been determined by taking the precision and accuracy of replicate measurements into account. The present study is focused on calibration processes (before, after, and during the mass measurement), the resolving power of the mass spectrometers, and the data processing for obtaining elemental formulae. The mass range between m/z 100 and 600 has been evaluated with a mix of four standards. This mass range includes small molecules usually detected in food and environmental samples. Negative ESI has been tested as there is almost no data on accurate mass (AM) measurements in this mode. Moreover, it has been used because it is the ESI mode for analysis of many compounds, such as pharmaceutical, herbicides, and fluorinated compounds. Natural organic matter has been used to demonstrate the significance of ultrahigh-resolution in complex mixtures. Sub-millidalton accuracy and precision have been obtained with Q-TOF, FT-ICR, and orbitrap achieving equivalent results. Poorer accuracy and precision have been obtained with the QqQ used: 11 mDa root-mean-square error and 6–11 mDa standard deviation. Some advice and requirements for daily AM routine analysis are also discussed here.     

Utility of three types of mass spectrometers for determining elemental compositions of ions formed from chromatographically separated compounds

Concentration factors of 1000 and more reveal dozens of compounds in extracts of water supplies. Library mass spectra for most of these compounds are not available, and alternative means of identification are needed. Determination of the elemental compositions of the ions in mass spectra makes feasible searches of commercial and chemical literature that often lead to compound identification. Instrumental capabilities that constrain the utility of a mass spectrometer for determining ion compositions for compounds that elute from a chromatographic column are scan speed, mass accuracy, linear dynamic range, and resolving power. Mass peak profiling from selected ion recording data (MPPSIRD) performed with a double-focusing mass spectrometer provides the best combination of these capabilities. This technique provides unique ion compositions for ions of higher mass from compounds eluting from a gas chromatograph than can be obtained by orthogonal acceleration time-of-flight (oa-TOF) or Fourier transform ion cyclotron resonance mass spectrometry. Multiple compositions are usually possible for an ion with a mass exceeding 150 Da within the error limits of the mass measurement. The correct composition is selected based on measured exact masses of the mass peak profiles resulting from isotopic ions higher in mass by 1 and 2 Da and accurate measurement of the summed abundances of these isotopic ions relative to the monoisotopic ion. A profile generation model (PGM) automatically determines which compositions are consistent with measured exact masses and relative abundances. The utility of oa-TOF and double-focusing mass spectrometry using ion composition elucidation (MPPSIRD plus the PGM) are considered for determining ion compositions of two compounds found in drinking water extracts and a third compound from a monitoring well at a landfill. Published in 2002 by John Wiley & Sons, Ltd.     

Evaluation of a High Resolving Power Time-of-Flight Mass Spectrometer for Drug Analysis in Terms of Resolving Power and Acquisition Rate

Liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) is applied increasingly to various fields of small molecule analysis. The moderate resolving power (RP) of standard TOFMS instruments poses a risk of false negative results when complex biological matrices are to be analyzed. In this study, the performance of a high resolving power TOFMS instrument (maXis by Bruker Daltonik, Bremen, Germany) was evaluated for drug analysis. By flow injection analysis of critical drug mixtures, including a total of 17 compounds with nominal masses of 212–415 Da and with mass differences of 8.8–23.5 mDa, RP varied from 34,400 to 51,900 (FWHM). The effect of acquisition rate on RP, mass accuracy, and isotopic pattern fit was studied by applying 1, 2, 5, 10, and 20 Hz acquisition rates in a 16 min gradient elution LC separation. All three variables were independent of the acquisition rate, with an average mass accuracy and isotopic pattern fit factor (mSigma) of 0.33 ppm and 5.9, respectively. The average relative standard deviation of RP was 1.8%, showing high repeatability. The performance was tested further with authentic urine extracts containing a co-eluting compound pair with a nominal mass of 296 Da and an 11.2 mDa mass difference. The authentic sample components were readily resolved and correctly identified by the automated data analysis. The average RP, mass accuracy, and isotopic pattern fit were 36,600, 0.9 ppm, and 7.3 mSigma, respectively.     

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.     

An Improved Measurement of Isotopic Ratios by High Resolution Mass Spectrometry

The study of protein kinetics requires an accurate measurement of isotopic ratios of peptides. Although Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometers yield accurate mass measurements of analytes, the isotopologue ratios are consistently lower than predicted. Recently, we demonstrated that the magnitude of the spectral error in the FT-ICR mass spectrometer is proportional to the scan duration of ions. Here, we present a novel isotopic ratio extrapolation (IRE) method for obtaining accurate isotopic ratio measurements. Accuracy is achieved by performing scans with different duration and extrapolation of the data to the initial moment of the ion rotation; IRE minimizes the absolute isotopic ratio error to ≤1 %. We demonstrate the application of IRE in protein turnover studies using ²H₂O-metabolic labeling. Overall, this technique allows accurate measurements of the isotopic ratios of proteolytic peptides, a critical step for enabling routine studies of proteome dynamics.      

Advanced Mass Calibration and Visualization for FT-ICR Mass Spectrometry Imaging

Mass spectrometry imaging by Fourier transform ion cyclotron resonance (FT-ICR) yields hundreds of unique peaks, many of which cannot be resolved by lower performance mass spectrometers. The high mass accuracy and high mass resolving power allow confident identification of small molecules and lipids directly from biological tissue sections. Here, calibration strategies for FT-ICR MS imaging were investigated. Sub-parts-per-million mass accuracy is demonstrated over an entire tissue section. Ion abundance fluctuations are corrected by addition of total and relative ion abundances for a root-mean-square error of 0.158?ppm on 16,764 peaks. A new approach for visualization of FT-ICR MS imaging data at high resolution is presented. The ??Mosaic Datacube?? provides a flexible means to visualize the entire mass range at a mass spectral bin width of 0.001?Da. The high resolution Mosaic Datacube resolves spectral features not visible at lower bin widths, while retaining the high mass accuracy from the calibration methods discussed.     

Performance evaluation of a high-field orbitrap mass analyzer

A new design of the Orbitrap^™ mass analyzer is presented. Higher frequencies of ion oscillations and hence higher resolving power over fixed acquisition time are achieved by decreasing the gap between the inner and outer Orbitrap electrodes, thus providing higher field strength for a given voltage. Experimental results confirm maximum FWHM resolving power in excess of 350,000 at m/z 524 and 600,000 at m/z 195, isotopic resolution of proteins above 40 kDa, and a single-shot dynamic range of 25,000. It was also found that mass shifts in the new design depend very little on space charge inside the analyzer. This performance was achieved using higher voltages and by careful balancing of construction tolerances and operation parameters, which appeared to vary in narrower ranges of tuning than for a standard Orbitrap analyzer.     

Using a triple-quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds

Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1 Da and +2 Da. When both properties are measured, a mass error limit of 6-10 mDa (< 31 ppm at 320 Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320 Da in this study). 'Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are -2, -1, +1, or +2 Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1 Da and +2 Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches.     

Resolving the composition of protein complexes using a MALDI LTQ orbitrap

Current biological studies have been advanced by the continuous development of robust, accurate, and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the Orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae, Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification compared with our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy, and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that (1) the resolving power, inversely proportional to the square root of m/z, had over four orders of magnitude dynamic range; (2) internal calibration led to improved accuracy, with an average absolute mass error of 0. 5 ppm and a distribution centered at 0 ppm; and (3) subfemtomole sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomole level of detection using neutral loss scanning.     

Complete large-molecule high-resolution mass spectra from 50-femtomole microvolume injection

For electrospray ionization in Fourier-transform mass spectrometry, direct injection of 5×10^?14 mol (0.5 µL of 100 nM from a microvolume sample valve) of ubiquitin (8565 Da) into the flowing solvent stream yields a spectrum with 85:1 signal-to-noise ratio, 2-ppm mass accuracy, and isotopic resolution. Gated trapping for 100 µs from a 0.15-µL/min injection of 20-µM ubiquitin consumes 5×10^?18 mol, which produces a spectrum with 23:1 signal-to-noise ratio and τ;3×10⁵ resolving power.     

Comparison of electron ionization mass spectra of some organic compounds (MW < 200 Da) recorded on mass spectrometers of various types

Low-resolution electron ionization mass spectra recorded on various types of mass spectrometers (time-of-flight, quadrupole, and three-dimensional ion trap) were compared. A model mixture of 10 organic compounds (MW < 200 Da) was analyzed by gas chromatography-mass spectrometry. Pure mass spectra of analytes were extracted using the AMDIS software. The best repeatability was achieved for the time-of-flight mass spectrometer. The mass spectra recorded by a quadrupole and a time-of-flight mass spectrometer were quite similar. In the case of these instruments, library search using a commercial mass spectral data base (NIST’05) gave satisfactory result for each analyte (rank 1 or 2 in the “hit list”; Match > 900). In some cases, the mass spectra of model compounds recorded by the ion trap mass spectrometer differed in intensity of certain mass spectral peaks (but not in the set of peaks) from the mass spectra presented in the library and from the experimental mass spectra recorded by the time-of-flight and quadrupole instruments.     

Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer

Herein, we represent a simple method for the detection and characterization of molecular species of triacylglycerol monohydroperoxides (TGOOH) in biological samples by use of reversed-phase liquid chromatography with a LTQ Orbitrap XL mass spectrometer (LC/LTQ Orbitrap) via an electrospray ionization source. Data were acquired using high-resolution, high-mass accuracy in Fourier-transform mode. Platform performance, related to the identification of TGOOH in human lipoproteins and plasma, was estimated using extracted ion chromatograms with mass tolerance windows of 5 ppm. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO₄ to generate oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No TGOOH molecular species were detected in the nLDL and nHDL, whereas 11 species of TGOOH molecules were detected in the oxLDL and oxHDL. In positive-ion mode, TGOOH was found as [M + NH₄]⁺. In negative-ion mode, TGOOH was observed as [M + CH₃COO]^–. TGOOH was more easily ionized in positive-ion mode than in negative-ion mode. The LC/LTQ Orbitrap method was applied to human plasma and three molecular species of TGOOH were detected. The limit of detection is 0.1 pmol (S/N?=?10:1) for each synthesized TGOOH.

Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control

Clinical and forensic toxicology and doping control deal with hundreds or thousands of drugs that may cause poisoning or are abused, are illicit, or are prohibited in sports. Rapid and reliable screening for all these compounds of different chemical and pharmaceutical nature, preferably in a single analytical method, is a substantial effort for analytical toxicologists. Combined chromatography–mass spectrometry techniques with standardised reference libraries have been most commonly used for the purpose. In the last ten years, the focus has shifted from gas chromatography–mass spectrometry to liquid chromatography–mass spectrometry, because of progress in instrument technology and partly because of the polarity and low volatility of many new relevant substances. High-resolution mass spectrometry (HRMS), which enables accurate mass measurement at high resolving power, has recently evolved to the stage that is rapidly causing a shift from unit-resolution, quadrupole-dominated instrumentation. The main HRMS techniques today are time-of-flight mass spectrometry and Orbitrap Fourier-transform mass spectrometry. Both techniques enable a range of different drug-screening strategies that essentially rely on measuring a compound’s or a fragment’s mass with sufficiently high accuracy that its elemental composition can be determined directly. Accurate mass and isotopic pattern acts as a filter for confirming the identity of a compound or even identification of an unknown. High mass resolution is essential for improving confidence in accurate mass results in the analysis of complex biological samples. This review discusses recent applications of HRMS in analytical toxicology.     

Thermal Expansion in the Zr and 1-, 2-valent Complex Phosphates of NaZr2(PO4)3 (NZP) Structure

A_5–4xZr_xZr(PO₄)₃ (A=Na, K;0≤x≤1.25), Na_1-xCd_0.5xZr₂(PO₄)₃ (0≤x≤1), Na_5–xCd_0.5xZr(PO₄)₃ (0≤x≤4) compositions which belong to the NZP structural family were synthesized using the sol-gel method. The lattice thermal expansion of members of these rows were determined up to 600°C by high-temperature X-ray diffractometry. The axial thermal expansion coefficients change from -5.8·10^-6to 7.5·10^-6 °C^-1 (α_a) and from 2.6·10^–6 to 22·10^–6 °C^-1 (α_c). These results, in addition to those for other NZP compounds allow us to explain their low thermal expansion. The mechanism can be attributed to strongly bonded three-dimensional network structure, the existence of structural holes capable to damp some of the thermal vibrations and anisotropyin the thermal expansion of the lattice. This revised version was published online in August 2006 with corrections to the Cover Date.