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.     

Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition

Labeling of the cell surface of Escherichia coli was accomplished by expression of a recombinant outer membrane protein, OmpC, in the presence of the unnatural amino acid azidohomoalanine, which acts as a methionine surrogate. The surface-exposed azide moieties of whole cells were biotinylated via Cu(1)-catalyzed [3+2] azide-alkyne cycloaddition. The specificity of labeling of both wild-type OmpC and a mutant containing additional methionine sites for azidohomoalanine incorporation was confirmed by Western blotting. Flow cytometry was performed to examine the specificity of the labeling. Cells that express the mutant form of OmpC in the presence of azidohomoalanine, which were biotinylated and stained with fluorescent avidin, exhibit a mean fluorescence 10-fold higher than the background. Incorporation of an unnatural amino acid can thus be determined on a single-cell basis.     

Adsorption of bovine alpha-lactalbumin on suspended solid nanospheres and its subsequent displacement studied by NMR spectroscopy

Detailed knowledge of the adsorption-induced conformational changes of proteins is essential to understand the process of protein adsorption. However, not much information about these conformational changes is available. Here, the adsorption of calcium-depleted (APO)- and calcium-containing (HOLO)-bovine alpha-lactalbumin (BLA) on suspended solid polystyrene nanospheres and their subsequent displacement by a surfactant are studied by NMR spectroscopy. To our knowledge, this is the first time that adsorption of proteins on solid nanospheres, with both components present in the NMR sample, is studied by this method. High-quality one-dimensional and two-dimensional 1H NMR spectra of nonadsorbed APO- and HOLO-BLA in the presence of BLA- and/or surfactant-covered solid polystyrene nanospheres in suspension are obtained using standard NMR procedures. BLA and surfactant molecules that are adsorbed on the polystyrene nanospheres give rise to extremely broadened proton resonances. This can be exploited to determine the amount of adsorbed protein and of adsorbed surfactant in a system containing protein, nanospheres, and surfactant, without disturbing the equilibrium of the system. Two-dimensional 1H NMR spectroscopy shows that the chemical shifts of the backbone amide protons of HOLO-BLA after its adsorption and subsequent displacement from polystyrene nanospheres by the surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) are identical to those of native HOLO-BLA. The adsorption-induced unfolding of BLA to a molten globule state on polystyrene nanospheres is thus fully reversible at the residue level upon CHAPS-induced displacement of BLA. The latter is the now fulfilled essential requirement that enables the future indirect study, at the residue level, of the conformational characteristics of BLA adsorbed on polystyrene nanospheres by hydrogen/deuterium exchange and NMR spectroscopy. The results presented show that NMR spectroscopy is clearly feasible to study the adsorption of BLA on suspended polystyrene nanospheres. This technique should be applicable to the study of the adsorption of other proteins on other surfaces as well.     

Highly selective asymmetric acetate aldol reactions of an N-acetyl thiazolidinethione reagent

[reaction: see text] A highly diastereoselective acetate aldol reaction that uses a tert-leucine-derived thiazolidinethione auxiliary and dichlorophenylborane has been developed. The reaction proceeds in excellent yields and with high diastereoselectivities (drs range from 9.5:1 to >100:1).     

Optimization of matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation using poly(ethylene glycol)

Details of the optimization of the collision-induced dissociation (CID) process, using a collision cell on a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer, are described using poly(ethylene glycol) 1000 (PEG 1000) as a model analyte. The effects of collision gas identity (helium, air, and argon), as well as collision gas pressure, on the resulting MS/MS data were investigated. With PEG 1000, helium was found to give the best results with respect to signal-to-noise (S/N) ratio. The optimum pressure for each gas was found to be in the range where the precursor ion signal was attenuated to approximately 30-50% for helium and 40-60% for argon. The effect of cation choice (Li, Na, and K) on the CID of PEG was also studied. CID spectra were produced for each, but PEG cationized with lithium was found to produce the spectra with the highest S/N ratio. The MALDI-TOF CID spectra that were generated for PEG were compared with the high-energy and low-energy MS/MS spectra obtained from a sector mass spectrometer and from a triple quadrupole mass spectrometer, respectively. The results observed for PEG confirm that CID on a MALDI-TOF mass spectrometer is a high-energy MS/MS technique.     

Solvent mediated interactions close to fluid-fluid phase separation: microscopic treatment of bridging in a soft-core fluid

Using density functional theory we calculate the density profiles of a binary solvent adsorbed around a pair of big solute particles. All species interact via repulsive Gaussian potentials. The solvent exhibits fluid-fluid phase separation, and for thermodynamic states near to coexistence the big particles can be surrounded by a thick adsorbed "wetting" film of the coexisting solvent phase. On reducing the separation between the two big particles we find there can be a "bridging" transition as the wetting films join to form a fluid bridge. The effective (solvent mediated) potential between the two big particles becomes long ranged and strongly attractive in the bridged configuration. Within our mean-field treatment the bridging transition results in a discontinuity in the solvent mediated force. We demonstrate that accounting for the phenomenon of bridging requires the presence of a nonzero bridge function in the correlations between the solute particles when our model fluid is described within a full mixture theory based upon the Ornstein-Zernike equations.     

Equilibrium of the reaction between dissolved sodium sulfide and biologically produced sulfur

The equilibrium of the heterogeneous reaction between dissolved sodium sulfide and biologically produced sulfur particles has been studied. Biologically produced sulfur was obtained from a bioreactor of a hydrogen sulfide removal process in which the dominating organism is Thiobacillus sp. W5. Detailed knowledge of this reaction is essential to understand its effect on the process. The results were compared with the equilibrium of the reaction of sulfide with ‘inorganic’ elemental sulfur. The equilibrium between dissolved sodium sulfide and biologically produced sulfur particles can be described by an equilibrium constant, K_x, which consists of a weighted sum of constants for polysulfide ions of different chain length, rather than a true single equilibrium constant. For biologically produced sulfur pK_x = 9.10 ± 0.08 (21 °C) and 9.17 ± 0.09 (35 °C) with an average polysulfide chain length x = 4.91 ± 0.32 (21 °C) and 4.59 ± 0.31 (35 °C). The pK_x value for biologically produced sulfur is significantly higher than for reaction of dissolved sodium sulfide with inorganic sulfur (pK_x = 8.82; 21 °C). This difference is probably caused by the negatively charged polymeric organic layer, which is present on biologically produced sulfur but absent with “inorganic” sulfur. Specific binding of polysulfide ions to the organic layer results in a higher polysulfide concentration at the reaction site compared to the bulk concentration. This results in an apparent decrease of the measured equilibrium constant, K_x.     

A study of [Co2(alkyne)(binap)(CO)4] complexes (BINAP=(1,1'-binaphthalene)-2,2'-diylbis(diphenylphosphine))

Understanding the interaction of chiral ligands, alkynes, and alkenes with cobaltcarbonyl sources is critical to learning more about the mechanism of the catalytic, asymmetric Pauson-Khand reaction. We have successfully characterized complexes of the type [Co2(alkyne)(binap)(CO)4] (BINAP=(1,1'-binaphthalene)-2,2'-diylbis(diphenylphosphine)) and shown that diastereomer interconversion occurs under Pauson-Khand reaction conditions when alkyne=HC[triple bond]CCO2Me. Attempts to isolate [Co2(alkyne)(binap)(CO)x] complexes with coordinated alkenes led to the formation of cobaltacyclopentadiene species.     

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.