Detection of protein-RNA crosslinks by nanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments

Protein-RNA interactions within ribonucleoprotein particles (RNPs) can be investigated by UV-induced crosslinking of proteins to their cognate RNAs and subsequent isolation and mass-spectrometric analysis of crosslinked peptide-RNA oligonucleotides. Because of the low crosslinking yield, a major challenge in protein-RNA UV crosslinking is the detection of the crosslinked species over the excess of non-crosslinked material, especially when complex systems (native RNPs) are investigated. Here, we applied a novel approach that uses on-line nanoLC-ESI-MS/MS to detect and subsequently sequence peptide-RNA oligonucleotide crosslinks from crude mixtures. To detect the crosslinks we made use of features shared by crosslinks and phosphopeptides, that is, the phosphate groups that both carry. A precursor ion scan for m/z 79 (negative-ion mode, -ve) is applied to selectively detect analytes bearing the phosphate-containing species (i.e., residual non-crosslinked RNA and peptide-RNA crosslinks) from crude mixtures and to determine their exact m/z values. On this basis, a multiple reaction monitoring (MRM) experiment monitors the expected decomposition from the different precursor charge states of the putative crosslinks to one of the four possible RNA nucleobases [m/z 112, 113, 136, 152 (positive-ion mode, +ve)]. On detection, a high-quality MS/MS is triggered to establish the structure of the crosslink. In a feasibility study, we detected and subsequently sequenced peptide-RNA crosslinks obtained by UV-irradiation of (1) native U1 snRNPs and (2) [15.5K-61K-U4atac] snRNPs prepared by reconstitution in vitro. MRM-triggered collision-induced dissociation (CID) MS/MS enabled us to obtain sequence information about the crosslinked peptide and RNA moiety.     

Assignment and extracting dynamics from experimentally and theoretically obtained spectroscopic hamiltonians in the complex spectral and classically chaotic regions

An analysis of existing algebraic multiresonance spectroscopic Hamiltonians, derived by fitting to experimental data or from classical canonical or quantum Van Vleck perturbation theory, allows without any significant further classical or quantum calculation the assignment of quantum numbers and motions to states observed in spectra that were previously thought to be irregular or just unassignable. In such cases, inspection of the amplitude and phase of eigenfunctions previously calculated in the Hamiltonians derivation process but now transformed to a reduced dimension semiclassical action-angle representation reveals extremely simple albeit unfamiliar topologies that give quantum numbers by simply counting nodes and phase advances. The topology allows these simple wave functions to be sorted into dynamically different excitation ladders or classes of states which are associated with different regions of phase space. The rungs of these ladders or the states in the classes intersperse in energy causing the spectral complexity. No experimental procedure allows such sorting. The success of the work stems from (1) the qualitative insights of nonlinear dynamics, (2) the conversion of the quantum problem in full dimension to a semiclassical one in reduced dimension by use of a canonical transform that takes advantage of the polyad and other constants of the motion, and (3) the judicious choice of the reduced angle variables to reflect rational ratio resonance frequency conditions. This leads to localization of those semiclassical wave functions, which are affected by the particular resonance. In reverse, the localized appearance of the reduced dimension wave function reveals which resonances govern it and makes sorting visually simple. The success of the work also stems from (4) the revealing use of plots of phase advances as well as the usual densities of the eigenstates for sorting and assignment purposes. Even in classically chaotic regions, organizing trajectories, which correspond to averages over regional phase space structures that need not be computed, can easily be drawn as the structure about which eigenfunction localization takes place. The organizing trajectories when transformed back to the full dimensional configuration space reveal the internal molecular motions. The complexity of the usual quantum stationary and propagated wave functions and associated classical trajectories forbids most often such assignments and sorting. This procedure brings the ability to interpret complex vibrational spectra to a degree previously thought possible only for lower excitations. The new methodology replaces and extends the computationally more difficult parts of a procedure used by the authors that was applied successfully to several molecules during the past few years. The new methodology is applied to DCO, CHBrClF, and the bending of acetylene.     

Site-specific labeling of DNA-protein conjugates by means of expressed protein ligation

Site-specific bioconjugation of protein thioesters with a DNA oligonucleotide was achieved by Expressed Protein Ligation (EPL) and the new thiol group formed upon EPL in the conjugate was selectively coupled with small molecule labels using maleimide chemistry.     

Isolation of a New Carotenoid and Two New Carotenoid Glycosides from Curtobacterium flaccumfaciens pvar poinsettiae

An efficient method for the extraction of the carotenoids from Curtobacterium flaccumfaciens pvar poinsettiae was developed. The glucosides of C.p. 450 (=(all‐E,2R,2′R)‐2‐[4‐(β‐D ‐glucopyranosyloxy)‐3‐methylbut‐2‐enyl]‐2′‐(4‐hydroxy‐3‐methylbut‐2‐enyl)‐β, β‐carotene; 4 ) and of C.p. 473 (=(all‐E,2R,2′S)‐2‐[4‐(β‐D ‐glucopyranosyloxy)‐3‐methylbut‐2‐enyl]‐2′‐(3‐methylbut‐2‐enyl)‐3′,4′‐didehydro‐1′,2′‐dihydro‐β,ψ‐caroten‐1′‐ol; 5 ) were isolated for the first time. In addition, the hitherto unknown 3′,4′‐dihydro derivative of C.p. 450, called C.p. 460 (=(all‐E,2R,2′R)‐2‐(4‐hydroxy‐3‐methylbut‐2‐enyl)‐2′‐(3‐methylbut‐2‐enyl)‐1′,2′‐dihydro‐β,ψ‐caroten‐1′‐ol; 6 ), was identified. The structures were established by UV/VIS, CD, ¹H‐ and ¹³C‐NMR, and mass spectra.     

Herstellung organischer Dünnstschichten

In this paper we present different experiments describing the fabrication and characterisation of self-assembled monolayers of organothiols and also discuss some of the most important applications of these systems. The reader may practise the fabrication and characterisation of self-assembled monolayers with different endgroups by adsorption of CH₃- and Oh-terminated alkanethiols on copper-coins (1 or 2 Pfenningcoins). Then patterned self-assembled monolayers of CH₃- and OH-terminated alkanethiols are produced by applying the microcontact printing technique. When these gratings are wet with water-e.g. by breathing into the surfaces-the water adsorbs onto the hydrophilic OH-terminated areas but not on the hydrophobic CH₃-terminated regions. Illumination with a laser then produces bright diffraction spots which disappear when the water evaporates. Finally, we briefly discuss applications of the self-assembled ultrathin films as sensor-devices and as substrates for CVD (chemical vapour deposition).     

DNA Microarrays

The complete human genes (ca. 100 000) as well as the whole spectrum of biological diversity should soon be able to be analyzed simultaneously by means of DNA microarrays using the fast technical advances that are occurring in this area. The particular strength of array analysis, typically based on the hybridization of nucleic acid probes attached to microchips with labeled RNA or DNA samples, results from the highly redundant measurement of many parallel hybridization events (see picture), which leads to an extraordinary level of assay validation.     

Investigation of airborne nanopowder agglomerate stability in an orifice under various differential pressure conditions

The stability of agglomerates is not only an important material parameter of powders but also of interest for estimating the particle size upon accidental release into the atmosphere. This is especially important when the size of primary particles is well below the agglomerate size, which is usually the case when the size of primary particles is below 100 nm. During production or airborne transportation in pipes, high particle concentrations lead to particle coagulation and the formation of agglomerates in a size range of up to some micrometers. Binding between the primary particles in the agglomerates is usually due to van der Waals forces. In the case of a leak in a pressurized vessel (e.g. reactor, transport pipe, etc.), these agglomerates can be emitted and shear forces within the leak can cause agglomerates to breakup. In order to simulate such shear forces and study their effect on agglomerate stability within the airborne state, a method was developed where agglomerate powders can be aerosolized and passed through an orifice under various differential pressure conditions. First results show that a higher differential pressure across the orifice causes a stronger fragmentation of the agglomerates, which furthermore seems to be material dependent.