Ion-Ion and Ion-Molecule Reactions at the Surface of Proteins Produced by Nanospray. Information on the Number of Acidic Residues and Control of the Number of Ionized Acidic and Basic Residues

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 μM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1–10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an nMAX which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A− form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution

Structural and dynamical characteristics of mesoscopic H+^+[H2_2O]n_n clusters

Structural and dynamical characteristics pertaining to the solvation of an excess proton in liquid-like nanoclusters of the type [H$_2$O]$_n$ are investigated using Molecular Dynamics experiments. Three different aggregate sizes were analyzed: $n=10,$ 21 and 125. The simulation experiments were performed using a multistate empirical valence bond Hamiltonian model. While in the smallest aggregates the proton occupies a central position, the stable solvation environments for $n=21$ and 125 are located at the cluster boundaries. In all cases, the structure of the closest solvation shell of the excess charge remains practically unchanged and coincides with that observed in bulk water. Compared to results obtained in bulk, the computed rates for proton transfer in clusters are between one and two orders of magnitude slower, and tend to increase for larger cluster sizes.Comment: 16 pages, 6 figures, to be published in Journal of Molecular Liquids, EMLG2006 special issu

On the suitability of high vacuum electrospray deposition for the fabrication of molecular electronic devices

We present a series of three studies investigating the potential application of high vacuum electrospray deposition to construct molecular electronic devices. Through the use of time of flight secondary ion mass spectrometry we explore the use of this novel deposition technique to fabricating multilayer structures using materials that are compatible with the same solvents and films containing a mixture of molecules from orthogonal solvents. Using x-ray photoelectron spectroscopy we study the deposition of a polymer blend using electrospray and find evidence of preferential deposition of one of the components

Collision of millimetre droplets induces DNA and protein transfection into cells

Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake

Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-tovacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental setup and optimization strategy is described for liquid AP-MALDI MS which improves the ionization effi- ciency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2 fmol/lL (0.5 lL, i.e. 1 fmol, deposited on the target) with very low sample consumption in the low nL-range

Current perspectives on supercharging reagents in electrospray ionization mass spectrometry

In electrospray ionization mass spectrometry (ESI-MS), analytes are introduced into the mass spectrometer in typically aqueous-organic solvent mixtures, including pH modifiers. One mechanism for improving the signal intensity and simultaneously increasing the generation of higher charge-state ions is the inclusion of small amounts (approx. <0.5% v/v mobile phase solution) of charge-inducing or supercharging reagents, such as m-nitrobenzyl alcohol, o-nitrobenzyl alcohol, m-nitrobenzonitrile, m-(trifluoromethyl)-benzyl alcohol and sulfolane. We explore the direct and indirect (colligative properties) that have been proposed as responsible for their modes of action during ESI. Of the many theorized mechanisms of ESI, we re-visit the three most popular and highlight how they are impacted by supercharging observations on small ions to large molecules including proteins. We then provide a comprehensive list of 34 supercharging reagents that have been demonstrated in ESI experiments. We include an additional 19 potential candidate isomers as supercharging reagents and comment on their broad physico-chemical properties. It is becoming increasingly obvious that advances in technology and improved ion source design, analyzers e.g. the use of ion mobility, ion trap, circular dichroism (CD) spectroscopy, together with computer modeling are increasing the knowledge base and, together with the untested isomers and yet-to-be unearthed ones, offer opportunities for further research and application in other areas of polymer research

Analysis of large oxygenated and nitrated polycyclic aromatic hydrocarbons formed under simulated diesel engine exhaust conditions (by compound fingerprints with SPE/LC-API-MS)

The analysis of organic compounds in combustion exhaust particles and the chemical transformation of soot by nitrogen oxides are key aspects of assessment and mitigation of the climate and health effects of aerosol emissions from fossil fuel combustion and biomass burning. In this study we present experimental and analytical techniques for efficient investigation of oxygenated and nitrated derivatives of large polycyclic aromatic hydrocarbons (PAHs), which can be regarded as well-defined soot model substances. For coronene and hexabenzocoronene exposed to nitrogen dioxide under simulated diesel exhaust conditions, several reaction products with high molecular mass could be characterized by liquid chromatography-atmospheric pressure chemical (and photo) ionization-mass spectrometry (LC-APCI-MS and LC-APPI-MS). The main products of coronene contained odd numbers of nitrogen atoms (m/z 282, 256, 338), whereas one of the main products of hexabenzocoronene exhibited an even number of nitrogen atoms (m/z 391). Various reaction products containing carbonyl and nitro groups could be tentatively identified by combining chromatographic and mass spectrometric information, and changes of their relative abundance were observed to depend on the reaction conditions. This analytical strategy should highlight a relatively young technique for the characterization of various soot-contained, semi-volatile, and semi-polar reaction products of large PAHs