Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Mapping and sequencing of cardiolipins from Geobacillus stearothermophilus NRS 2004/3a by positive and negative ion nanoESI-QTOF-MS and MS/MS

In the course of systematic studies on surface layer (S-layer) glycoproteins of bacilli, the chloroform/methanol extract from whole cells of Geobacillus stearothermophilus NRS 2004/3a has been submitted to MS analysis. Glucosylated cardiolipins were found as minor components of the total lipid and phospholipid mixture by de novo identification. After purification of the crude extract using a combined column chromatography/2D TLC protocol, structural investigations of components in the lipid fraction by high resolution ESI-QTOF MS analysis provided evidence about homologous molecules attributable to the cardiolipin species containing a glycosylated backbone, and about a diversity of ester-linked fatty acid substituents. In comparative studies by positive and negative ion nanoESI-QTOF-CID-MS, maps of cardiolipin molecular ions were obtained, followed by MS/MS of the most abundant species, to provide structural details of D-glucopyranosylcardiolipin and the fatty acid substituent patterns. Experiments of the parent ion scan type revealed the presence of fatty acid moieties as isobaric combinations, represented in single molecular ion species.     

Sheathless reverse-polarity capillary electrophoresis-electrospray-mass spectrometry for analysis of underivatized glycoconjugates

We report on the development of a novel methodology to extend the limits of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) general applicability. A sheathless on-line CE-ESI-MS setup was optimized on standard monosaccharide mixture to operate in reverse polarity and negative ion mode for MS detection without pressure to assist the sample migration, coating of the capillaries, and/or sample derivatization. This approach was further applied for screening of a complex glycopeptide mixture obtained from the urine of a patient diagnosed with N-acetylhexosaminidase deficiency, known as Schindler's disease. The potential of this methodology in terms of high sensitivity, separation efficiency, resolution, and reproducibility is demonstrated. In combination with the high quality of MS data, a new, significantly improved insight into the sample heterogeneity is possible.     

An interpolation theorem for proper holomorphic embeddings

Given a Stein manifold x of dimension n > 1, a discrete sequence , and a discrete sequence where , there exists a proper holomorphic embedding satisfying f(a _j ) = b _j for every j = 1,2,... Forstnerič and Prezelj supported by grants P1-0291 and J1-6173, Republic of Slovenia. Kutzschebauch supported by Schweizerische National fonds grant 200021-107477/1. Ivarsson supported by The Wenner-Gren Foundations.     

Switching time in optically bistable injection-locked semiconductor lasers

Switching between states in a dispersive bistable injection-locked slave laser has been theoretically investigated. We show that the switching can be achieved by relatively small and short (≈10-50?ps) variation of the master laser injection power or frequency, which, besides the variation of the slave laser optical power, leads to significant variation of its photon phase (≈5π/6). By using an analytical model, we calculate the switching time dependence on the magnitude of the injection power and the frequency detuning variation.     

Variations of trace element contents within single tree rings

X-ray fluorescence was used to measure variations in relative and absolute trace-element concentrations within single tree rings taken from a 32-year-old pine tree near a coalburning power plant. The Zn/Cu ratio was found to have a constant value, both along individual rings and between different rings. The Fe, Ca and 8 concentrations varied in a single tree ring and depended on the age of the tree. The results indicate that the variations in concentrations of elements along a single ring are caused by heterogeneous tree tissue structure.     

Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 15 degrees C and 25 degrees C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Lipooligosaccharides (LOSs) are macromolecules present on the external cellular membrane of Gram-negative bacteria, structurally made of two distinct regions, lipid A and Core. By varying their growth temperature, bacteria such as psychrophiles change the phosphorylation distribution of the LOSs produced. The level of phosphorylation and the phosphate group positions in LOSs produced by the extremophile psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125, grown at 15 degrees C and 25 degrees C, were investigated by nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) and tandem mass spectrometry (MS/MS). The samples, obtained by phenol/chloroform/petroleum ether (PCP) extraction of dried cells, were treated with hydrazine at 37 degrees C in order to reduce the heterogeneity by removal of the ester-linked fatty acid moieties. The molecular ion distributions in these LOS fractions were investigated in negative ion mode. Based on these data it was postulated that the sample grown at 25 degrees C contained four phosphate groups while that at 15 degrees C contained three. In order to determine phosphorylation sites in sugar chains, the samples were submitted to low collision energy MS/MS for sequencing. In the sample with three phosphates, one was found to be linked to the tetrasaccharide Core region, more precisely to position C-4 of the Kdo unit. The two remaining phosphate groups were both linked to the 2-acylamide-2-deoxy-D-glucopyranose of the lipid A moiety, and two possible distributions could be postulated on the basis of the fragmentation pattern obtained; in the first case both phosphate groups are linked as a pyrophosphate moiety to position C-1 of the proximal glucosamine (reducing residue), while in the second case one phosphate is linked to position C-1 of the proximal glucosamine and the other to position C-4' of the distal glucosamine (non-reducing residue). This distribution was also found in the lipid A moiety of the tetraphosphorylated sample grown at 25 degrees C, which bears two phosphate groups on the Core region, one on position C-4 of the Kdo and the other on position C-7 or C-8 of the same residue. The phosphate locations were derived from the intra-ring cleavage ions of sugar moieties in the LOSs obtained by an optimized CID procedure using negative ion QTOF-MS/MS.     

Structures involving unshared electron pair. The Crystal Structures of As(OCOCH3)3 and As2O(OCOCH3)4

As₂O(OCOCH₃)₄, reported now for the first time, was obtained, besides As(OCOCH₃)₃ by dissolving As₂O₃ in acetic anhydride. The crystals of As(OCOCH₃)₃ (A) (monoclinic, space group Cc, Z = 4, a = 9.970(2), b = 13.203(2), c = 8.272(1) Å, β = 117.01(2)°) and of As₂O(OCOCH₃)₄ (B) (monoclinic, space group P2₁/n, Z = 4, a = 13.966(5), b = 8.127(4), c = 12.706(4) Å, β = 95.14(1)°) are built up from discrete molecules defined by chemical formulae As(OCOCH₃)₃ and (CH₃OCO)₂As? O? As(OCOCH₃)₂, respectively. The molecular structure of both compounds is based on the AsO₃ pyramid: in (A) with the As? O bonds of 1.841(6) Å and the O? As? O angle of 89.9(3)° as a mean, in (B) with slightly different values and with the As? O? As angle of 127.7(4)° at the bridging oxygen atom. The additional weak chelating contacts are at the distances As…O from 2.625(9) to 2.745(10) Å in (A) and from 2.72(1) to 2.84(2) in (B). The actual arsenic coordination can be described as very distorted octahedral in (A) and square-pyramidal in (B).     

Chemical Characterization of Airborne Particles in St. Martinus Cathedral in Weert, The Netherlands

Electron probe X-ray microanalysis (EPMA) of single particles and energy dispersive X-ray fluorescence analysis (EDXRF) were applied to determine the chemical composition, size and probable origin of the suspended particulate matter. The aim of the performed research was to determine the chemical composition, size and abundance of aerosol particles responsible for blackening and soiling of the works of art displayed within the Cathedral of Weert in the Netherlands and to verify the possible sources responsible for these processes.