The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument

The effect of elevating the pressure in the interface region of an electrospray ionization orthogonal time-of-flight mass spectrometer on the ion intensity of different noncovalent protein assemblies has been investigated. Elevating the pressure in the interface region generally led to an enhanced detection of high m/z ions. The optimum pressure was found to be dependent on the m/z value of the ions. This pressure effect should be carefully addressed when relating ion abundance in the mass spectra to solution phase abundance of noncovalent protein assemblies.     

Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination

In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are systematically varied. Our findings suggest that, under the experimental conditions we use, the extent of intramolecular hydrogen scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier, and therefore most likely by the gas-phase structure of the peptide ion. Moreover, the observed scrambling seems to be independent of the nature of the peptide fragment ions (i.e., protonated B and Y' ' ions, and sodiated A and Y' ions). Our results strongly suggest that scrambling may be reduced by using alkali metal cationization instead of protonation in the ionization process.     

Recent liquid chromatographic-(tandem) mass spectrometric applications in proteomics

Conventional proteomics makes use of two-dimensional gel electrophoresis followed by mass spectrometric analysis of tryptic fragments derived from in-gel digestion of proteins. Although being a very strong technique capable of separating and visualizing hundreds of proteins, 2D-gel electrophoresis has some well-documented disadvantages as well. More recently, liquid chromatographic-(tandem) mass spectrometric techniques have been developed to overcome some of the shortcomings of 2D-gel electrophoresis. In this review we have described several recent applications of liquid chromatography-(tandem) mass spectrometry in the field of proteomics and especially in the field of membrane proteomics, quantitative proteomics and in the analysis of post-translational modifications.     

Carbohydrate chain of ganglioside GM1 as a ligand: identification of the binding strategies of three 15 mer peptides and their divergence from the binding modes of growth-regulatory galectin-1 and cholera toxin

The branched pentasaccharide chain of ganglioside GM1 is a prominent cell surface ligand, for example, for cholera toxin or tumor growth-regulatory homodimeric galectins. This activity profile via protein recognition prompted us to examine the binding properties of peptides with this specificity. Our study provides insights into the mechanism of molecular interaction of this thus far unexplored size limit of the protein part. We used three pentadecapeptides in a combined approach of mass spectrometry, NMR spectroscopy and molecular modelling to analyze the ligand binding in solution. Availability of charged and hydrophobic functionalities affected the intramolecular flexibility of the peptides differently. Backfolding led to restrictions in two cases; the flexibility was not reduced significantly by association of the ligand in its energetically privileged conformations. Major contributions to the interaction energy arise from the sialic acid moiety contacting Arg/Lys residues and the N-terminal charge. Considerable involvement of stacking between the monovalent ligand and aromatic rings could not be detected. This carbohydrate binding strategy is similar to how an adenoviral fiber knob targets sialylated glycans. Rational manipulation for an affinity enhancement can now be directed to reduce the flexibility, exploit the potential for stacking and acquire the cross-linking capacity of the natural lectins by peptide attachment to a suitable scaffold.     

Analysis of Early Medieval Glass Beads – The Raw Materials to Produce Green, Orange and Brown Colours

 Monochrome coloured glass beads of the Merovingians (5^th–7^th cent. AD) have been examined by different analytical methods. The elemental composition of a large number of mostly unprepared beads have been measured non-destructively by X-ray fluorescence analysis. After subtracting the content of the colouring oxides of the glass beads and normalising the residual values to 100% an identical soda-lime-glass matrix was obtained. X-ray diffraction was used for the identification of the crystalline colouring and opacifying pigments (SnO₂, Cu, Cu₂O, PbSnO₃), and scanning electron microscopy as well as electron probe microanalysis were applied to study the microstructure and the composition of white, brown, green, orange and yellow coloured glass beads. Oxidised metals, alloys (lead, copper, bronze, brass and mixtures of them) and iron smelting slag have been identified as raw materials to colour the soda-lime-glass.     

A new symmetrically modified α-cyclodextrin tripode: selective metal complexation and fluorescence properties

A new tripodal α-cyclodextrin having three ureido-bipyridyl tethers symmetrically distributed on its upper rim was prepared in one step by the polymer supported ‘phosphine imide’ reaction in a medium yield. As expected, the highly selective complexation properties towards ‘hard’ and ‘soft’ cations were maintained with regards to the URFT-Cd (upper rim fully tethered) family previously investigated. Its Eu^III and Tb^III lanthanide complex fluorescence behaviour clearly indicates that a limited number of tethers allow a better bis-heterocyclic antenna self-organisation around the cation leading to enhanced fluorescence properties.     

Zinc solid-state NMR spectroscopy of human carbonic anhydrase: implications for the enzymatic mechanism

The pH dependence of the (67)Zn solid-state nuclear magnetic resonance spectroscopy of human carbonic anhydrase (CAII) has been investigated to characterize the nature of the fourth ligand. CAII, through the Zn(2+)-bound hydroxide, catalyzes the deceptively simple reaction: CO(2) + H(2)O <==> HCO(3)(-) + H(+). The accepted mechanism for CAII would predict that water would be bound to the Zn(2+) at pH 5 and hydroxide would be bound at pH 8.5. The measured values for the electric field gradient (EFG) or quadrupole coupling constant (Cq) for CAII are independent of pH within the limits of the experimental error, i.e., 9.8 +/- 0.2 MHz. The EFG interaction has been predicted by ab initio electronic structure calculations for water and hydroxide bound to the zinc, including various levels of hydrogen bonding. After comparing the predicted Cq's with the experimental values, we conclude that the species present from pH 5-8.5 is the hydroxide form. The NMR data presented here is not consistent with the accepted mechanism for CAII. We show that the NMR data is consistent with an alternative mechanism of CAII.