Advances in the analysis of dynamic protein complexes by proteomics and data processing

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.     

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Post-translational modifications of proteins are involved in determining the activity of proteins and are essential for proper protein function. Current mass spectrometric strategies require one to specify a particular type of modification, in some cases also a particular charge state of a protein or peptide that is to be studied before the actual analysis. Due to these requirements, most of the modifications on proteins are not considered in such an experiment and, thus, a series of similar analyses need to be performed to ensure a more extensive characterization. A novel scan strategy has been developed, multiple neutral loss monitoring (MNM), allowing for the comprehensive screening of post-translational modifications (PTM) on proteins that fragment as neutral losses in a mass spectrometer. MNM method parameters were determined by performing product ion scans on a number of modified peptides over a range of collision energies, providing neutral loss energy profiles and optimal collision energies (OCE) for each modification, supplying valuable information pertaining to the fragmentation of these modifications and the necessary parameters that would be required to obtain the best analysis. As the optimal collision energy was highly dependent on the type of modification and the charge state of the peptide, the MNM scan was operated with a collision energy gradient. Autocorrelation analyses identified the type of modification, and convolution mapping analyses identified the associated peptide. The MNM scan with the new collision energy parameters was successfully applied to a mixture of four modified peptides in a BSA digest. The implementation of this technique will allow for comprehensive screening of all modifications that fragment as neutral losses.     

Synthesis,Modification, and Biological Evaluation of a Library of Novel Water-Soluble Thiopyridone-Based Organometallic Complexes and Their Unexpected (Biological) Behavior

A series of 16 dinuclear thiopyridone-based organometallics with excellent water solubility, increased stability and remarkable cytotoxicity were synthesized and characterized. The complexes of this work formed dimeric species featuring a double positive charge in polar protic solvents, accounting for their outstanding solubility in aqueous solution. Most of them displayed higher antiproliferative activity than their parental thiomaltol complex, with unexpected cytotoxicity trends depending on the employed metal center, ligand modification, and cell line. Insights into their behavior in biological systems were gathered by means of amino-acid interaction studies, cytotoxicity tests in 3D spheroid models, laser ablation, cellular accumulation measurements, as well as cell cycle experiments.     

Atmospheric pressure-electron capture dissociation of peptides using a modified PhotoSpray ion source

An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévote's group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.     

Delayed gamma rays from muon capture on 207Pb

We have measured the energies and intensities of delayed γ-rays from ^207,201Tl following muon capture on ²⁰⁷Pb. Probabilities for the emission of 0–6 neutrons have been deduced. Measured and predicted neutron multiplicities (based on an impulse approximation model) are in reasonable agreement for multiplicities of 2, 3, 4 and 5. Experimental values for high and low multiplicities are not accounted for by the theory. High angular momentum states involving the $\text{h}{}_{\mathrm{<mtext>11</mtext><mtext>2</mtext>}}$ proton hole are observed in several of the thallium daughters. Using a simple shell-model approach, we have correlated μ-capture on the $\text{h}{}_{\mathrm{<mtext>11</mtext><mtext>2</mtext>}}$ proton shell with the energy spectrum of directly emitted neutrons.     

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Ewald summation versus direct summation of shifted-force potentials for the calculation of electrostatic interactions in solids: A quantitative study

The calculated Madelung energies and Madelung forces of the electrostatic interaction for nine crystal structures are reported. The method of direct summation with two different shifted-force potentials is compared to the Ewald summation. There is a considerable difference in the convergence of the energy and the force for the two shifted-force potentials regarding the cutoff radius. The convergence depends not only on the potential itself, but also on the crystal structure. One of the shifted-force potentials used is implemented in the CHARMM force field. The energy calculated with this potential shows a good convergence for small cutoff radii. With the other shifted-force potential, the force shows a better convergence for small cutoff radii. The number of pair interactions for obtaining the Madelung limit using the Ewald summation and the direct summation of a shifted-force potential is also reported. For complex structures like zeolites, the number of relevant pair interactions is smaller using the direct summation of a shifted-force potential. For simple structures such as cesium chloride, the number of significant pair interactions is smaller using the Ewald summation. © 1997 by John Wiley & Sons, Inc.     

Coincidence Doppler shift lifetime measurements in 73,74Se and 74Br using the EUROBALL Cluster cube array

Subpicosecond lifetimes of high spin states in the rotational nuclei ⁷³Se, ⁷⁴Se and ⁷⁴Br have been measured using the ⁵⁸Ni +¹⁹F compound reaction and the Doppler Shift Attenuation method. Six EUROBALL Cluster detectors arranged in cube geometry allowed us to select the relevant transitions in γγ coincidence mode. The high counting statistics achieved in this setup also facilitated the determination of average sidefeeding times, which were found to agree rather well with the results of Monte-Carlo calculations of the particle and γ-ray evaporation process. The deduced quadrupole strengths and deformations are compared with the results of previous measurements and the predictions of Cranked Shell Model calculations. In ⁷⁴Br, a large and constant prolate deformation of β₂= 0.37(1) was found for the presumed 4⁺ and 3⁻ two-quasiparticle bands. Received: 2 July 1999     

Magnetic moment measurements in the semi-magic nuclei 94Ru and 95Rh after recoil implantation into iron and nickel

The magnetic moments of the 12⁺ and 11⁻ yrast states in ⁹⁴Ru and of the 25/2⁻, 29/2⁺, and 35/2⁺ levels in ⁹⁵Rh have been measured via the IMPAD technique. The nuclei were produced in the reaction ⁵⁸Ni +⁴⁰Ca and recoil-implanted into polarized Ni and Fe hosts. The g-factors were deduced from the measured time-integral Larmor precessions. The comparison between the experimental results and large-scale shell model calculations suggests that the 12⁺ and 11⁻ states in ⁹⁴Ru and the 25/2⁻ level in ⁹⁵Rh are pure proton states whereas the 29/2⁺ and 35/2⁺ states in ⁹⁵Rh contain a neutron excitation across the N=50 shell gap. This interpretation supports the conclusion drawn from recent lifetime measurements. Received: 2 June 1999