Time-Resolved Ion Mobility Spectrometry with a Stop Flow Confined Volume Reaction Region.

An ion source concept is described where the sample flow is stopped in a confined volume of an ion mobility spectrometer creating time-dependent patterns of ion patterns of signal intensities for ions from mixtures of volatile organic compounds and improved signal-to-noise rate compared to conventional unidirectional drift gas flow. Hydrated protons from a corona discharge were introduced continuously into the confined volume with the sample in air at ambient pressure, and product ions were extracted continuously using an electric field for subsequent mobility analysis. Ion signal intensities for protonated monomers and proton bound dimers were measured and computationally extracted using mobilities from mobility spectra and exhibited distinct times of appearance over 30 s or more after sample injection. Models, and experimental findings with a ternary mixture, suggest that the separation of vapors as ions over time was consistent with differences in the reaction rate for reactions between primary ions from hydrated protons and constituents and from cross-reactions that follow the initial step of ionization. The findings suggest that the concept of stopped flow, introduced here for the first time, may provide a method for the temporal separation of atmospheric pressure ions. This separation relies on ion kinetics and does not require chromatographic technology.

Enzymatic Phosphorylation of Oxidized Tyrosine Residues.

Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of specific regulating actions of enzymes, such as tyrosine kinases phosphorylating tyrosine residues or by nonenzymatic reactions, such as oxidation related to oxidative stress and diseases. While many studies have addressed the multisite, dynamic, and network-like properties of PTMs, only little is known of the interplay of the same site modifications. In this work, we studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which tyrosine residues were replaced with l-DOPA. The phosphorylated peptides were identified by liquid chromatography-high-resolution mass spectrometry and the site of phosphorylation by tandem mass spectrometry. The results clearly show that the oxidized tyrosine residues are phosphorylated, displaying a specific immonium ion peak in the MS2 spectra. Furthermore, we detected this modification in our reanalysis (MassIVE ID: MSV000090106) of published bottom-up phosphoproteomics data. The modification, where both oxidation and phosphorylation take place at the same amino acid, has not yet been published in PTM databases. Our data indicate that there can be multiple PTMs that do not exclude each other at the same modification site.

Mechanism of the Oxidation of Heptafulvenes to Tropones Studied by Online Mass Spectrometry and Density Functional Theory Calculations.

We have identified the most likely reaction mechanism for oxidizing heptafulvenes to the corresponding tropones by experimental and theoretical investigations. The experimental studies were done by coupling a three-dimensional printed miniaturized reactor with an integrated electrospray ionization needle to a mass spectrometer. Using the experimentally observed ions as a basis, nine alternative reaction pathways were investigated with density functional theory calculations. The lowest energy reaction pathway starts with the formation of an epoxide that is opened upon the addition of a second equivalent of the oxidizing species meta-chloroperoxybenzoic acid. The adduct formed then undergoes a Criegee-like rearrangement to yield a positively charged hemiketal, which on deprotonation dissociates into acetone and tropone. Overall, the reaction mechanism resembles a Hock-like rearrangement.

TiO2 Photocatalysis-DESI-MS Rotating Array Platform for High-Throughput Investigation of Oxidation Reactions.

We present a new high-throughput platform for studying titanium dioxide (TiO2) photocatalytic oxidation reactions by performing reactions on a TiO2-coated surface, followed by direct analysis of oxidation products from the surface by desorption electrospray ionization mass spectrometry (DESI-MS). For this purpose, we coated a round glass wafer with photocatalytically active anatase-phase TiO2 using atomic layer deposition. Approximately 70 aqueous 1 µL samples can be injected onto the rim of the TiO2-coated glass wafer, before the entire wafer is exposed to UV irradiation. After evaporation of water, the oxidation products can be directly analyzed from the sample spots by DESI-MS, using a commercial rotating sample platform. The method was shown to provide fast photocatalytic oxidation reactions and analysis with throughput of about four samples per minute. The feasibility of the method was examined for mimicking phase I metabolism reactions of amodiaquine, buspirone and verapamil. Their main photocatalytic reaction products were mostly similar to the products observed earlier in TiO2 photocatalysis and in in vitro phase I metabolism assays performed using human liver microsomes.

Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis.

Protein phosphorylation has a key role in cell regulation. Oxidation of proteins, in turn, is related to many diseases and to aging, but the effects of phosphorylation on the oxidation of proteins and peptides have been rarely studied. The aim of this study was to examine the mechanistic effect of phosphorylation on peptide oxidation induced by titanium dioxide photocatalysis. The effect of phosphorylation was compared between nonphosphorylated and tyrosine phosphorylated peptides using electrospray tandem mass spectrometry. We observed that tyrosine was the most preferentially oxidized amino acid, but the oxidation reaction was significantly inhibited by its phosphorylation. The study also shows that titanium dioxide photocatalysis provides a fast and easy method to study oxidation reactions of biomolecules, such as peptides.

Shape-anchored porous polymer monoliths for integrated online solid-phase extraction-microchip electrophoresis-electrospray ionization mass spectrometry.

We report a simple protocol for fabrication of shape-anchored porous polymer monoliths (PPMs) for on-chip SPE prior to online microchip electrophoresis (ME) separation and on-chip (ESI/MS). The chip design comprises a standard ME separation channel with simple cross injector and a fully integrated ESI emitter featuring coaxial sheath liquid channel. The monolith zone was prepared in situ at the injection cross by laser-initiated photopolymerization through the microchip cover layer. The use of high-power laser allowed not only maskless patterning of a precisely defined monolith zone, but also faster exposure time (here, 7 min) compared with flood exposure UV lamps. The size of the monolith pattern was defined by the diameter of the laser output (∅500 µm) and the porosity was geared toward high through-flow to allow electrokinetic actuation and thus avoid coupling to external pumps. Placing the monolith at the injection cross enabled firm anchoring based on its cross-shape so that no surface premodification with anchoring linkers was needed. In addition, sample loading and subsequent injection (elution) to the separation channel could be performed similar to standard ME setup. As a result, 15- to 23-fold enrichment factors were obtained already at loading (preconcentration) times as short as 25 s without sacrificing the throughput of ME analysis. The performance of the SPE-ME-ESI/MS chip was repeatable within 3.1% and 11.5% RSD (n = 3) in terms of migration time and peak height, respectively, and linear correlation was observed between the loading time and peak area.

Desorption atmospheric pressure photoionization high-resolution mass spectrometry: a complementary approach for the chemical analysis of atmospheric aerosols.

RATIONALE: On-line chemical characterization methods of atmospheric aerosols are essential to increase our understanding of physicochemical processes in the atmosphere, and to study biosphere-atmosphere interactions. Several techniques, including aerosol mass spectrometry, are nowadays available, but they all suffer from some disadvantages. In this research, desorption atmospheric pressure photoionization high-resolution (Orbitrap) mass spectrometry (DAPPI-HRMS) is introduced as a complementary technique for the fast analysis of aerosol chemical composition without the need for sample preparation. METHODS: Atmospheric aerosols from city air were collected on a filter, desorbed in a DAPPI source with a hot stream of toluene and nitrogen, and ionized using a vacuum ultraviolet lamp at atmospheric pressure. To study the applicability of the technique for ambient aerosol analysis, several samples were collected onto filters and analyzed, with the focus being on selected organic acids. To compare the DAPPI-HRMS data with results obtained by an established method, each filter sample was divided into two equal parts, and the second half of the filter was extracted and analyzed by liquid chromatography/mass spectrometry (LC/MS). RESULTS: The DAPPI results agreed with the measured aerosol particle number. In addition to the targeted acids, the LC/MS and DAPPI-HRMS methods were found to detect different compounds, thus providing complementary information about the aerosol samples. CONCLUSIONS: DAPPI-HRMS showed several important oxidation products of terpenes, and numerous compounds were tentatively identified. Thanks to the soft ionization, high mass resolution, fast analysis, simplicity and on-line applicability, the proposed methodology has high potential in the field of atmospheric research.

Computational analysis of an electrostatic separator design for removal of volatile organic compounds from indoor air.

Concentrations of volatile organic compounds (VOCs) in air can be reduced in electrostatic separators where VOCs are ionized using ion-molecule reactions, extracted using electric fields, and eliminated in a waste flow. Embodiments for such separator technology have been explored in only a few studies, despite the possible advantage of purification without adsorbent filters. In one design, based on ionization of VOCs in positive polarity with hydrated protons as reactant ions, efficiencies for removal were measured as 30-40% . The results were fitted to a one-dimensional convective diffusion model requiring an unexpectedly high production rate of reactant ions to match both the model and data. A realistic rate of reactant ion production was used in finite element method simulations (COMSOL) and demonstrated that low removal efficiency could be attributed to non-uniform patterns of sample flow and to incomplete mixing of VOCs with reactant ions. In analysis of complex systems, such as this model, even limited computational modeling can outperform a pure analytical approach and bring insights into limiting factors or system bottlenecks.Implications: In this work, we applied modern computational methods to understand the performance of an air purifier based on electrostatics and ionized volatile organic compounds (VOCs). These were described in the publication early 2000s. The model presented was one-dimensional and did not account for the effects of flow. In our multiphysics finite element models, the efficiency and operation of the filter is better explained by the patterns of flow and flow influences on ion distributions in electric fields. In general, this work helps using and applying computational modelling to understand and improve the performance bottlenecks in air purification system designs.

Quantitative Distributions of Product Ions and Reaction Times with a Binary Mixture of VOCs in Ambient Pressure Chemical Ionization.

A model to quantitatively predict ion abundances from atmospheric pressure chemical ionization (APCI) between hydrated protons and a volatile organic compound (VOC) was extended to binary mixtures of VOCs. The model includes differences in vapor concentrations, rate coefficients, and reaction times and is enhanced with cross reactions between neutral vapors and protonated monomers. In this model, two specific VOCs were considered, a ketone, 6-methyl-5-hepten-2-one (M, and an amine, 2,6-di-tert-butyl-pyridine (N), with measured "conditional rate coefficients" (in cm3·s-1) of kM = 1.11 × 10-9 and kN = 9.17 × 10-10, respectively. The cross reaction of MH+(H2O)x to NH+(H2O)y was measured as kcr = 1.31 × 10-12 at 60 °C. Cross reactions showed an impact on ion abundances at t > 30 ms for equal vapor concentrations of 100 ppb for M and N. In contrast, this impact was negligible for vapor concentrations of 1 ppb and did not exceed 5% change in product ion abundance up to 1000 ms reaction times. The model was validated with laboratory measurements to within ∼10% using an ion mobility spectrometer and effective reaction time obtained from computational fitting of experimental findings. This was necessitated by complex flow patterns in the ion source volume and was determined as ∼10.5 ms. The model has interpretative and predictive value for quantitative analysis of responses with ambient pressure ion sources for mass spectrometry and ion mobility spectrometry.

Microchip technology in mass spectrometry.

Microfabrication of analytical devices is currently of growing interest and many microfabricated instruments have also entered the field of mass spectrometry (MS). Various (atmospheric pressure) ion sources as well as mass analyzers have been developed exploiting microfabrication techniques. The most common approach thus far has been the miniaturization of the electrospray ion source and its integration with various separation and sampling units. Other ionization techniques, mainly atmospheric pressure chemical ionization and photoionization, have also been subject to miniaturization, though they have not attracted as much attention. Likewise, all common types of mass analyzers have been realized by microfabrication and, in most cases, successfully applied to MS analysis in conjunction with on-chip ionization. This review summarizes the latest achievements in the field of microfabricated ion sources and mass analyzers. Representative applications are reviewed focusing on the development of fully microfabricated systems where ion sources or analyzers are integrated with microfluidic separation devices or microfabricated pums and detectors, respectively. Also the main microfabrication methods, with their possibilities and constraints, are briefly discussed together with the most commonly used materials.

Letter: A simple ion source set-up for desorption/ionization on silicon with ion mobility spectrometry and ion mobility spectrometry-mass spectrometry.

Using a simple ion source set-up, laser desorption/ionization on silicon (DIOS) was demonstrated with the use of a custom-made drift tube ion mobility spectrometer (IMS), mounted on a commercial triple quadrupole mass spectrometer, and with an IMS equipped with a Faraday plate detector. DIOS was tested by mobility measurement of tetrapropylammonium iodide, tetrabutylammonium iodide and tetrapentylammonium iodide, whilst 2,6-di-tert- butylpyridine was used as a standard. The reduced mobilities measured for the test halides are in concordance with previously obtained ion mobility spectrometry-mass spectrometry data.

Ion density of positive and negative ions at ambient pressure in air at 12-136 mm from 4.9 kV soft x-ray source.

The abundance of ions is an essential parameter for ion mobility and mass spectrometry instrument design and for the control or optimization of chemical reactions with reactant ions. This information also advances the study of atmospheric pressure ion kinetics under continuous ionization, which has a role in developing trace level chemical analyzers. In this study, an ionization chamber is described to measure the abundance of ions produced by a 4.9 keV, model L12535, soft x-ray source from Hamamatsu Corporation. Ions of positive and negative polarity were measured independently in an 8 × 30 mm2 cross section at distances of 12-136 mm at ambient air from an uncollimated beam. Ions were collected using electric fields and 16 sets of plates. The ion current decreased exponentially with distance from the source, and the calculated ion concentration varied between 1.0 × 108 and 3.8 × 105 ions cm-3 on plates. A 2D-COMSOL model including losses by recombination and diffusion was favorably matched to changes in ion current intensity in the ionization chamber. Although the ionization chamber was built to characterize a commercial ion source, the design may be considered generally applicable to other x-ray sources.

Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry.

We developed a new multiplexed reversed phase liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) method. The method is based on isobaric labeling with a tandem mass tag (TMT10-plex) and stable isotope-labeled internal standards, and was used to analyze amino acids in mouse brain microdialysis samples. The TMT10-plex labeling of amino acids allowed analysis of ten samples in one LC-MS/MS run, significantly increasing the sample throughput. The method provides good chromatographic performance (peak half-width between 0.04-0.12 min), allowing separation of all TMT-labeled amino acids with acceptable resolution and high sensitivity (limits of detection typically around 10 nM). The use of stable isotope-labeled internal standards, together with TMT10-plex labeling, ensured good repeatability (relative standard deviation ≤ 12.1 %) and linearity (correlation coefficient > 0.994), indicating good quantitative performance of the multiplexed method. The method was applied to study the effect of d-amphetamine microdialysis perfusion on amino acid concentrations in the mouse brain. All amino acids were reliably detected and quantified, indicating that the method is sensitive enough to detect low concentrations of amino acids in brain microdialysis samples.

Parametric Sensitivity in a Generalized Model for Atmospheric Pressure Chemical Ionization Reactions.

Gas phase reactions between hydrated protons H+(H2O)n and a substance M, as seen in atmospheric pressure chemical ionization (APCI) with mass spectrometry (MS) and ion mobility spectrometry (IMS), were modeled computationally using initial amounts of [M] and [H+(H2O)n], rate constants k1 to form protonated monomer (MH+(H2O)x) and k2 to form proton bound dimer (M2H+(H2O)z), and diffusion constants. At 1 × 1010 cm-3 (0.4 ppb) for [H+(H2O)n] and vapor concentrations for M from 10 ppb to 10 ppm, a maximum signal was reached at 4.5 µs to 4.6 ms for MH+(H2O)x and 7.8 µs to 46 ms for M2H+(H2O)z. Maximum yield for protonated monomer for a reaction time of 1 ms was ∼40% for k1 from 10-11 to 10-8 cm3·s-1, for k2/k1 = 0.8, and specific values of [M]. This model demonstrates that ion distributions could be shifted from [M2H+(H2O)z] to [MH+(H2O)x] using excessive levels of [H+(H2O)n], even for [M] > 10 ppb, as commonly found in APCI MS and IMS measurements. Ion losses by collisions on surfaces were insignificant with losses of <0.5% for protonated monomer and <0.1% for proton bound dimer of dimethyl methylphosphonate (DMMP) at 5 ms. In this model, ion production in an APCI environment is treated over ranges of parameters important in mass spectrometric measurements. The models establish a foundation for detailed computations on response with mixtures of neutral substances.

Fabrication of nanocluster silicon surface with electric discharge and the application in desorption/ionization on silicon-mass spectrometry.

This study presents a new, simple, and low-cost technique to fabricate a nanocluster silicon (NCSi) surface on planar silicon using a micro-scale direct current (DC) discharge under ambient conditions. The method requires no masks, chemicals, vacuum environment, or laser, but only a high-voltage supply. The NCSi surfaces, characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy, consist of oxidized silicon nanoclusters 50-200 nm in diameter, likely formed by melting due to high temperatures in the discharge. The minimum size of the NCSi spot is determined by the size of the discharge tip (approximately 90 microm). Arbitrary NCSi areas can be produced on a silicon wafer by moving the discharge needle on the surface with the help of a computer-controlled xyz stage. NCSi surfaces can also be formed on three-dimensional (3D) surfaces, as demonstrated with silicon micropillars. NCSi surfaces can be used, for example, in various analytical applications. In this study, we demonstrate their use as sample plates in the analysis of drugs and peptides with desorption/ionization on silicon-mass spectrometry (DIOS-MS).

Hybrid ceramic polymers: new, nonbiofouling, and optically transparent materials for microfluidics.

A new, commercial hybrid ceramic polymer, Ormocomp, was introduced to the fabrication of microfluidic separation chips using two independent techniques, UV lithography and UV embossing. Both fabrication methods provided Ormocomp chips with stable cathodic electroosmotic flow which enabled examination of the Ormocomp biocompatibility by means of microchip capillary electrophoresis (MCE) and (intrinsic) fluorescence detection. The hydrophobic/hydrophilic properties of Ormocomp were examined by screening its interactions with bovine serum albumin and selected amino acids of varying hydrophobicity. The results show that the ceramic, organic-inorganic polymer structure natively resists biofouling on microchannel walls even so that the Ormocomp microchips can be used in intact protein analysis without prior surface modification. With theoretical separation plates approaching 10(4) m(-1) for intact proteins and 10(6) m(-1) for amino acids and peptides, our results suggest that Ormocomp microchips hold record-breaking performance as microfluidic separation platforms. In addition, Ormocomp was shown to be suitable for optical fluorescence detection even at near-UV range (ex 355 nm) with detection limits at a nanomolar level ( approximately 200 nM) for selected inherently fluorescent pharmaceuticals.

Dynamic coating of SU-8 microfluidic chips with phospholipid disks.

In this work, PEG-stabilized phosphatidylcholine lipid aggregates (disks), mimicking mammalian cell membranes, were introduced as a new biofouling resistant coating for SU-8 polymer microchannels. A rapid and simple method was developed for immobilization of PEGylated phosphatidylcholine disks in microchannels. Microfluidic chips made from SU-8, PDMS, or glass were dynamically coated with the PEGylated disks followed by characterization of their surface chemistry before and after coating. On the basis of the observed changes in EOF and nonspecific protein adsorption, the affinity of the PEGylated disks was shown to be particularly strong toward SU-8. The PEG-lipid coating enabled permanent change in EOF in SU-8 microchannels with an initial value of 4.5 x 10(-8) m(2) V(-1) s(-1), decreasing to 2.1 x 10(-8) m(2) V(-1) s(-1) (immediately after modification), and, eventually, to 1.5 x 10(-8) m(2) V(-1) s(-1) (7 days after modification) for 9 mM sodium borate (pH 10.5) as BGE. As determined by the Wilhelmy plate measurements and microchip-CE analysis of BSA, the PEG-lipid coating also enabled efficient biofouling shield against protein adsorption, similar to that of low amounts of SDS (3.5 mM) or Tween-20 (80 microM) as buffer additives. These results suggest that dynamically attached PEG-lipid aggregates provide stable, biomimicking surface modification that efficiently reduces biofouling on SU-8.

Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates.

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, ß-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.

Ionspray microchip.

An ionspray microchip is introduced. The chip is based on the earlier presented nebulizer microchip that consists of glass and silicon plates bonded together. A liquid inlet channel, nebulizer gas inlet, and nozzle are etched on the silicon plate and a platinum heater is integrated on the glass plate. The nebulizer microchip has been previously used in atmospheric pressure chemical ionization, atmospheric pressure photoionization, sonic spray ionization, and thermospray ionization modes. In this work we show that the microchip can be operated also in ionspray mode by introducing high voltage to the silicon plate of the microchip. The effects of operation parameters (voltage, nebulizer gas pressure, sample solution flow rate, solvent composition, and analyte concentration) on the performance of the ion spray microchip were studied. Under optimized conditions the microchip provides efficient ionization of small and large compounds and good quantitative performance. The feasibility of the ion spray microchip in liquid chromatography/mass spectrometry (LC/MS) was demonstrated by the analysis of tryptic peptides of bovine serum albumin.

Feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization mass spectrometry in analyzing anabolic steroids in urine samples.

We examined the feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization tandem mass spectrometry (capLC/microAPPI-MS/MS) for the analysis of anabolic steroids in human urine. The urine samples were pretreated by enzymatic hydrolysis (with beta-glucuronidase from Helix pomatia), and the compounds were liquid-liquid extracted with diethyl ether. After separation the compounds were vaporized by microchip APPI, photoionized by a 10 eV krypton discharge lamp, and detected by selected reaction monitoring. The capLC/microAPPI-MS/MS method showed good sensitivity with detection limits at the level of 1.0 ng mL(-1), good linearity with correlation coefficients between 0.9954 and 0.9990, and good repeatability with relative standard deviations below 10%. These results demonstrate that microchip APPI combined with capLC/MS/MS provides a new potential method for analyzing non-polar and neutral compounds in biological samples.