Ion Elevators and Escalators in Multilevel Structures for Lossless Ion Manipulations.

We describe two approaches based upon ion "elevator" and "escalator" components that allow moving ions to different levels in structures for lossless ion manipulations (SLIM). Guided by ion motion simulations, we designed elevator and escalator components based upon ion current measurements providing essentially lossless transmission in multilevel designs. The ion elevator design allowed ions to efficiently bridge a 4 mm gap between levels. The component was integrated in a SLIM and coupled to a QTOF mass spectrometer using an ion funnel interface to evaluate the m/z range transmitted as compared to transmission within a level (e.g., in a linear section). The analysis of singly charged ions of m/z 600-2700 produced similar mass spectra for both elevator and straight (linear motion) components. In the ion escalator design, traveling waves (TW) were utilized to transport ions efficiently between two SLIM levels. Ion current measurements and ion mobility (IM) spectrometry analysis illustrated that ions can be transported between TW-SLIM levels with no significant loss of either ions or IM resolution. These developments provide a path for the development of multilevel designs providing, e.g., much longer IM path lengths, more compact designs, and the implementation of much more complex SLIM devices in which, e.g., different levels may operate at different temperatures or with different gases.

Resistive-Pulse Analysis of Single Phospholipid Vesicles Using Quartz Nanochannels.

We report the use of resistive-pulse method and quartz nanochannels for the detection and size analysis of single vesicles. Cylindrical shape quartz nanochannels have been used to detect single phospholipid vesicles ranging from 100 to 300 nm and polystyrene nanoparticles ranging from 170 to 400 nm in diameter. Translocations of single vesicles and nanoparticle were detected as individual square current pulses, which could be used to determine particle size. Our results show excellent agreement between the particle/vesicle sizes obtained from nanochannels and those from dynamic light scattering (DLS) and scanning electron microscopy (SEM). This electronic-based method was found to be fast, simple, and used cheap and robust microsensors made in house. The application of a quartz channel might be combined with other analytical methods, such as amperometry and fluorescence microscopy, to yield more complete information about biological and artificial vesicles.

Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay.

Despite recent advancements in large-scale phosphoproteomics, methods to quantify kinase-specific phosphorylation stoichiometry of protein substrates are lacking. We developed a method to quantify kinase-specific phosphorylation stoichiometry by combining the reverse in-gel kinase assay (RIKA) with high-resolution liquid chromatography-mass spectrometry (LC-MS). Beginning with predetermined ratios of phosphorylated to nonphosphorylated protein kinase CK2 (CK2) substrate molecules, we employed 18O-labeled adenosine triphosphate (18O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of 18O- versus 16O-labeled tryptic phosphopeptide using high mass accuracy mass spectrometry (MS). We demonstrate that the phosphorylation stoichiometry determined by this method across a broad percent phosphorylation range correlated extremely well with the predicted value (correlation coefficient = 0.99). This approach provides a quantitative alternative to antibody-based methods of determining the extent of phosphorylation of a substrate pool.

A fundamental study on the [(µ-Cl)3Mg2(THF)6]+ dimer electrolytes for rechargeable Mg batteries.

The long sought solvated [MgCl](+) species in the Mg-dimer electrolytes was characterized by soft mass spectrometry. The presented study provides an insightful understanding on the electrolyte chemistry of rechargeable Mg batteries.

On the ionization and ion transmission efficiencies of different ESI-MS interfaces.

The achievable sensitivity of electrospray ionization mass spectrometry (ESI-MS) is largely determined by the ionization efficiency in the ESI source and ion transmission efficiency through the ESI-MS interface. These performance characteristics are difficult to evaluate and compare across multiple platforms as it is difficult to correlate electrical current measurements to actual analyte ions reaching the detector of a mass spectrometer. We present an effective method to evaluate the overall ion utilization efficiency of an ESI-MS interface by measuring the total gas-phase ion current transmitted through the interface and correlating it to the observed ion abundance measured in the corresponding mass spectrum. Using this method, we systematically studied the ion transmission and ionization efficiencies of different ESI-MS interface configurations, including a single emitter/single inlet capillary, single emitter/multi-inlet capillary, and a subambient pressure ionization with nanoelectrospray (SPIN) MS interface with a single emitter and an emitter array, respectively. Our experimental results indicate that the overall ion utilization efficiency of SPIN-MS interface configurations exceeds that of the inlet capillary-based ESI-MS interface configurations.

Mobility-resolved ion selection in uniform drift field ion mobility spectrometry/mass spectrometry: dynamic switching in structures for lossless ion manipulations.

A Structures for Lossless Ion Manipulations (SLIM) module that allows ion mobility separations and the switching of ions between alternative drift paths is described. The SLIM switch component has a "Tee" configuration and allows the efficient switching of ions between a linear path and a 90-degree bend. By controlling switching times, ions can be efficiently directed to an alternative channel as a function of their mobilities. In the initial evaluation the switch is used in a static mode and shown compatible with high performance ion mobility separations at 4 Torr. In the dynamic mode, we show that mobility-selected ions can be switched into the alternative channel, and that various ion species can be independently selected based on their mobilities for time-of-flight mass spectrometer (TOF MS) IMS detection and mass analysis. This development also provides the basis of, for example, the selection of specific mobilities for storage and accumulation, and the key component of modules for the assembly of SLIM devices enabling much more complex sequences of ion manipulations.

High sensitivity combined with extended structural coverage of labile compounds via nanoelectrospray ionization at subambient pressures.

Subambient pressure ionization with nanoelectrospray (SPIN) has proven to be effective in producing ions with high efficiency and transmitting them to low pressures for increased sensitivity in mass spectrometry (MS) analysis. Here we present evidence that the SPIN source not only improves MS sensitivity but also facilitates the detection of more labile compounds. The gentleness of conventional heated capillary electrospray ionization (ESI) and the SPIN designs was compared in conjunction with the liquid chromatography mass spectrometry (LC-MS) analysis of colominic acid and N-glycans containing sialic acid. Prior experiments conducted with the SPIN interface demonstrated the ability to detect labile glycans such as heavily sialylated and polysialic acid N-glycans, which are difficult to detect with a conventional ESI-MS interface. Colominic acid is a mixture of sialic acid polymers of different lengths containing labile glycosidic linkages between monomer units necessitating a gentle ion source. These labile covalent bonds may display similar behavior to sialic acid chains in N-glycans during MS analysis. By coupling the SPIN source with high-resolution mass spectrometry and using advanced data processing tools, we demonstrate much extended coverage of sialic acid polymer chains as compared to conventional ESI-MS and the ability to detect sialic acid containing N-glycans without the need of sample derivatization. In addition, we show that SPIN-LC-MS is effective in elucidating polymer features with high efficiency and high sensitivity previously unattainable by the conventional ESI-LC-MS methods.

Polysialylated N-glycans identified in human serum through combined developments in sample preparation, separations, and electrospray ionization-mass spectrometry.

The N-glycan diversity of human serum glycoproteins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesizable by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient, providing additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) was utilized. When these improved methods are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described (Kronewitter et al. Anal. Chem. 2014, 86, 6268-6276), we are able to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrated the application of these advances in the context of the human serum glycome, and for which our initial observations included the detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.

Improving the sensitivity of mass spectrometry by using a new sheath flow electrospray emitter array at subambient pressures.

Arrays of chemically etched emitters with individualized sheath gas capillaries were developed to enhance electrospray ionization (ESI) efficiency at subambient pressures. By incorporating the new emitter array in a subambient pressure ionization with nanoelectrospray (SPIN) source, both ionization efficiency and ion transmission efficiency were significantly increased, providing enhanced sensitivity in mass spectrometric analyses. The SPIN source eliminates the major ion losses of conventional ESI-mass spectrometry (MS) interfaces by placing the emitter in the first reduced pressure region of the instrument. The new ESI emitter array design developed in this study allows individualized sheath gas around each emitter in the array making it possible to generate an array of uniform and stable electrosprays in the subambient pressure (10 to 30 Torr) environment for the first time. The utility of the new emitter arrays was demonstrated by coupling the emitter array/SPIN source with a time of flight (TOF) mass spectrometer. The instrument sensitivity was compared under different ESI source and interface configurations including a standard atmospheric pressure single ESI emitter/heated capillary, single emitter/SPIN and multi-emitter/SPIN configurations using an equimolar solution of nine peptides. The highest instrument sensitivity was observed using the multi-emitter/SPIN configuration in which the sensitivity increased with the number of emitters in the array. Over an order of magnitude MS sensitivity improvement was achieved using multi-emitter/SPIN compared with using the standard atmospheric pressure single ESI emitter/heated capillary interface.

Redox-filled Carbon-Fiber Microelectrodes for Single-Cell Exocytosis.

Carbon-fiber microelectrodes (CFEs) are the primary electroanalytical tool in single-cell exocytosis and in-vivo studies. Here we report a new study on the kinetic properties of electrolyte-filled CFEs in single-cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)63- and Ru(NH3)63+, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)63- in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron-transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox-filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in-vivo sensing.

Steady-state voltammetry of a microelectrode in a closed bipolar cell.

Here we report the theory and experimental study of the steady-state voltammetric behavior of a microelectrode used as a limiting pole in a closed bipolar electrochemical cell. We show that the steady-state voltammetric response of a microelectrode used in a closed bipolar cell can be quantitatively understood by considering the responses of both poles in their respective conventional two-electrode setups. In comparison to a conventional electrochemical cell, the voltammetric response of the bipolar cell has a similar sigmoidal shape and limiting current; however, the response is often slower than that of the typical two-electrode setup. This leads to a broader voltammogram and a decreased wave slope, which can be somewhat misleading, causing the appearance that the process being studied is irreversible when it instead can be a result of the coupling of two reversible processes. We show that a large limiting current on the excess pole would facilitate the observation of a faster voltammetric response and that both redox concentration and electrode area of the excess pole affect the wave shape. Both factors should be maximized in electroanalytical experiments in order to obtain fast voltammetric responses on the main electrode of interest and to detect quick changes in analyte concentrations.

Nanoelectrodes: recent advances and new directions.

This article reviews recent work involving the development and application of nanoelectrodes in electrochemistry and related areas. We first discuss common analytical methods for characterizing the size, shape, and quality of nanoelectrodes, including electron microscopy, steady-state cyclic voltammetry, scanning electrochemical microscopy, and surface modification. We then emphasize recent developments in fabrication techniques that have led to structurally well-defined nanoelectrodes. We highlight recent advances in the application of nanoelectrodes in important analytical chemistry areas, such as single-molecule studies, single-nanoparticle electrochemistry, and measurements of neurotransmitters from single neuronal cells.

Electrochemical responses and electrocatalysis at single au nanoparticles.

Steady-state electrochemical responses have been obtained at single Au nanoparticles using Pt nanoelectrodes. A Au single-nanoparticle electrode (SNPE) is constructed by chemically immobilizing a single Au nanoparticle at a SiO(2)-encapsulated Pt disk nanoelectrode, which was previously modified by an amine-terminated silane. The Au SNPE has been characterized by transmission electron microscopy, underpotential deposition of Cu, and steady-state cyclic voltammetry. It has been found that the presence of a single Au nanoparticle enhances the electron transfer from the Pt nanoelectrode to the redox molecules, and the voltammetric response at the Au SNPE depends on the size of the Au nanoparticle. The Au SNPE has been utilized to examine the oxygen-reduction reaction in a KOH solution to explore the feasibility of measuring the electrocatalytic activity at a single-nanoparticle level. It has been shown that the electrocatalytic activity of single Au nanoparticles can be directly measured using SNPEs, and the electrocatalytic activity is dependent on the size of the Au nanoparticles. This study can help to understand the structure-function relationship in nanoparticle-based electrocatalysis.