Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions

The charge state distribution of proteins was studied as a function of experimental conditions, to improve the understanding of the matrix-assisted laser desorption/ionization (MALDI) mechanisms. The relative abundances of the multiply-charged ions appear to be a function of the matrix chosen, the laser fluence and the matrix-to-analyte molar ratio. A correlation is found between the matrix proton affinity and the yield of singly- versus multiply-charged ions. These results are in good agreement with a model in which gas-phase intracluster reactions play a significant role in analyte ion formation. A new model for endothermic desolvation processes in ultraviolet/MALDI is presented and discussed. It is based upon the existence of highly-charged precursor clusters and, complementary to the ion survivor model of Karas et al., assumes that two energy-dependent processes exist: (i) a soft desolvation involving consecutive losses of neutral matrix molecules, leading to a multiply-charged analyte and (ii) hard desolvation leading to a low charge state analyte, by consecutive losses of charged matrix molecules. These desolvations pathways are discussed in terms of kinetically limited processes. The efficiency of the two competitive desolvation processes seems related to the internal energy carried away by clusters during ablation.     

Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.     

Standardization for oxygen isotope ratio measurement - still an unsolved problem

Numerous organic and inorganic laboratory standards were gathered from nine European and North American laboratories and were analyzed for their delta(18)O values with a new on-line high temperature pyrolysis system that was calibrated using Vienna standard mean ocean water (VSMOW) and standard light Antartic precipitation (SLAP) internationally distributed reference water samples. Especially for organic materials, discrepancies between reported and measured values were high, ranging up to 2 per thousand. The reasons for these discrepancies are discussed and the need for an exact and reliable calibration of existing reference materials, as well as for the establishment of additional organic and inorganic reference materials is stressed. Copyright 1999 John Wiley & Sons, Ltd.     

Selective biomolecular nanoarrays for parallel single-molecule investigations

The ability to direct the self-assembly of biomolecules on surfaces with true nanoscale control is key for the creation of functional substrates. Herein we report the fabrication of nanoscale biomolecular arrays via selective self-assembly on nanopatterned surfaces and minimized nonspecific adsorption. We demonstrate that the platform developed allows for the simultaneous screening of specific protein-DNA binding events at the single-molecule level. The strategy presented here is generally applicable and enables high-throughput monitoring of biological activity in real time and with single-molecule resolution.     

Ab initio calculations of the photoionization of diatomic molecules

A review is presented of the calculation of photoionization spectra, particularly in the spectral range where electron autoionization of diatomic molecules takes place. In addition to some interesting results obtained over years that compare favourably with experiment, the emphasis here is put on the relation between the methods developed for the calculation of observables associated with the continuum energy spectrum of the electrons and the Alchemy system of programs. This system of programs serves as a basis for initial and intermediate calculations. The examples presented show that diatomic molecules not only in gas phase but also oriented in space or physisorbed at surfaces may be studied readily.     

The Influence of Alkali Metal Ions on the Stability and Reactivity of Chromium(III) Superoxide Moieties Spanned by Siloxide Ligands

In recent years, it has become clear that the presence of redox-inactive Lewis acidic metal ions can decisively influence the reactivity of metal–dioxygen moieties that are formed in the course of O₂ activation, in molecular complexes, and metalloenzymes. Superoxide species are often formed as the primary intermediates but they are mostly too unstable for a thorough investigation. We report here a series of chromium(III) superoxide complexes [L₂Cr]M₂O₂(THF)_y (L=⁻OSiPh₂OSiPh₂O⁻, M⁺=Li⁺, Na⁺, K⁺ and y=4, 5), which could be accessed, studied spectroscopically and partly crystallized at low temperatures. They only differ in the two incorporated Lewis acidic alkali metal counterions (M⁺) and it could thus be shown that the nature of M⁺ determines considerably its interaction with the superoxide ligand. This interaction, in turn, has a significant influence on the stability and reactivity of these complexes towards substrates with OH groups. Furthermore, we show that stability and reactivity are also highly solvent dependent (THF versus nitriles), as donor solvents coordinate to the alkali metal ions and thus also influence their interaction with the superoxide moiety. Altogether, these results provide a comprehensive and detailed picture concerning the correlation between spectroscopic properties, structure, and behavior of such superoxides, that may be exemplary for other systems.     

(1)H relaxation times of metabolites in biological samples obtained with nondestructive ex-vivo slow-MAS NMR

Methods suitable for measuring (1)H relaxation times such as T(1), T(2) and T(1rho) of metabolites in small, intact biological objects including live cells, excised organs and tissues, oil seeds etc. are developed in this work. This was achieved by combining inversion-recovery, spin-echo, or a spin-lock segment with the phase-adjusted spinning sideband (PASS) technique, which was applied at low sample-spinning rates. Here, PASS was used to produce high-resolution (1)H spectra in a nondestructive way so that the relaxation parameters of individual metabolite could be determined. The methodologies were demonstrated by measuring (1)H T(1), T(2), and T(1rho) of metabolites in excised rat liver at a spinning rate of 40 Hz.     

The effective dielectric constant of a dispersion of clustering spheres

If one measures the dielectric constant of a water in oil microemulsion the large increase of this dielectric constant both as function of the temperature as well as a function of the volume fraction of water suggests that the water droplets will bind together in clusters. In order to analyse this suggestion in more detail we derive a systematic expansion of the Clausius-Mossotti function in terms of integrals over products of excess cluster polarizabilities and correlation functions. It is found that the excess polarizability of a cluster of spherical droplets is zero except when the spheres almost touch each other. Crucial for this property is the fact that the water spheres are conducting or if they are not conducting have a dielectric constant much larger than the dielectric constant of oil. The large increase of the dielectric constant is a consequence of the increase of the number of bound clusters. An explicit formula is given for the resulting temperature and volume fraction dependence of the dielectric constant.Dedicated to Professor Harry Thomas on the occasion of his 60th birthday     

Optical properties of thin films on rough surfaces

A general theory, of second order in the film thickness and surface roughness over the wavelength, is developed of the optical properties of a thin film. These properties are described by a small number of electromagnetic constitutive coefficients. Formulae for these coefficients are derived in terms of the height-height correlation functions of the upper and lower surfaces of the film, and its average thickness. The reflectance, transmittance and ellipsometric coefficient are expressed in terms of the constitutive coefficients, for arbituary angles of incidence.     

Sensitivity-enhanced phase-corrected ultra-slow magic angle turning using multiple-echo data acquisition

The increase in the sensitivity of the phase-corrected magic angle turning (PHORMAT) experiment at ultra-slow spinning rates by means of multiple-echo data acquisition (ME-PHORMAT) is evaluated. This is achieved by replacing the acquisition dimension in the original experiment with a train of equally spaced pi-pulses. It is shown that the echoes following the odd and even pi-pulses in the CPMG train must be processed differently in order to avoid spectral distortions. The method is illustrated for 13C CP-ME-PHORMAT on solid 1,2,3-trimethoxybenzene and for 1H ME-PHORMAT on excised rat liver tissue, both at a sample-spinning rate of 1.3 Hz. Sensitivity enhancements of a factor 4 for the solid and 2.3 for the liver were obtained. Finally, it is shown that with ME-PHORMAT one of the two RF pulse sequences, in standard PHORMAT used to obtain a pure absorption mode 2D spectrum, can be eliminated, thus reducing the usually long measuring time by a factor 2.