Nanospray Capillary Research Articles

Development of Magnetic Molecularly Imprinted Polymer Coupled Nanospray Ion Source for Analysis of Cephalosporin Antibiotics in Food Samples.

A magnetic molecularly imprinted polymer (MMIP) coupled nanospray ion source was developed for analysis of cephalosporin antibiotics in food samples. MIP coated Fe3O4 nanospheres were prepared for magnetic solid-phase extraction (MSPE) of the antibiotics in the extract of samples and then integrated into the nanospray capillary for further desorption and mass spectrometry analysis. The developed device combines the advantages of high extraction efficiency of MSPE, unique selectivity of MIPs, and fast analysis speed of ambient ionization mass spectrometry (AIMS). Five cephalosporin antibiotics in milk, egg, and beef samples were analyzed using the developed methods. High sensitivities with limits of detection (LODs) from 0.3 to 0.5 μg kg-1 were achieved for cephalosporin antibiotics in milk, egg, and beef samples, respectively. Good linearity, determination coefficient values (R2 > 0.992), and precision (RSD < 15%) with recoveries ranging from 72.6% to 115.5% were obtained using the spiked milk, egg, and beef sample matrices.

Direct infusion nano-electrospray ionization mass spectrometry for therapeutic drug monitoring of ciprofloxacin and its metabolites in human saliva

A rapid and sensitive method based on direct infusion-nano-electrospray ionization mass spectrometry (DI-nESI-MS) has been developed for the detection and quantification of ciprofloxacin and its metabolites in human saliva. Saliva samples were collected after the oral administration of 500 mg ciprofloxacin tablets. Internal standard (IS), tamoxifen, was added to the collected samples, and then diluted with the ionization solvent, centrifuged and filtered. An aliquot of 4 μL of the filtrate was loaded into a nanospray (NS) capillary. The NS capillary was then fitted into an off-line ion source and the instrument was operated to acquire a two-minute run by applying a voltage of 1000 V (positive-ion detection mode). Quantification of ciprofloxacin relied on the ratio of its peak intensity to the IS peak intensity. The DI-nESI-MS method was validated and provided satisfactory precision with relative standard deviation ranging from 0.39 to 7.48 % and accuracy with relative error ranging from -2.12 to 9.72 %. The calibration curve showed good linearity (r2) > 0.999 over the concentration range of 10−4000 ng/mL. These results verify the effectiveness of the DI-nESI-MS method for monitoring of ciprofloxacin and its metabolites in human saliva samples.

Dilute-and-shoot-based direct nano-electrospray ionization tandem mass spectrometry as screening methodology for multivitamins in dietary supplement and human urine

IntroductionIn recent years, analytical screening methods for simultaneous detection of multivitamins have gained substantial attention to ensure quality and public confidence in dietary supplements. Even so, few analytical methods have been proposed for simultaneous analysis of multivitamin constituents due to the large divergence in chemical characteristics. ObjectivesIn the present study, the objective was to develop a simple and rapid direct nano-electrospray ionization-tandem mass spectrometry (DI-nano-ESI-MS/MS) method for targeted detection of water soluble vitamins, fat soluble vitamins, amino acids, royal jelly, ginkgo biloba, and ginseng in a dietary supplement. The applicability of dilute-and-shoot-based DI-nano-ESI-MS/MS to analyze the same tested compounds and their related metabolites in clinical samples was also examined. MethodsIntact urine mixed with the ionization solvent was loaded (4-μL aliquot) into a nanospray (NS) capillary of 1-μm tip diameter. The NS capillary was then fitted into an off-line ion source at a distance of 5 mm from MS aperture. The sample was directly injected by applying a voltage of 1.1 kV, producing a numerous of m/z peaks for analysis in mere minutes. ResultsThe DI-nano-ESI-MS/MS method successfully identified almost all dietary supplement components, as well as a plethora of component-related metabolites in clinical samples. In addition, a new merit of the proposed method for the detection of index marker and chemical contaminants as well as subspecies identification was investigated for further quality evaluation of the dietary supplement. ConclusionsThe previous findings illustrated that DI-nano-ESI-MS/MS approach can emerge as a powerful, high throughput, and promising analytical tool for screening and accurate detection of various pharmaceuticals and ingredient in dietary supplements as well as biological fluids.

Quantification and targeted detection of ciprofloxacin in dosage form and human urine by direct injection nano-electrospray ionization multi-stage mass spectrometry

A rapid, selective and sensitive direct infusion nano-electrospray ionization multi-stage mass spectrometry (DI-nano-ESI-MS3) method has been developed and validated for ciprofloxacin identification and quantification. An aliquot of 4 µl containing ciprofloxacin sample solution along with an internal standard (IS), in this case tamoxifen, was loaded into a nanospray (NS) capillary. The NS capillary was then fitted off-line into the ion source where the distance between NS capillary tip and MS aperture was set to 5 mm. The instrument was operated to acquire a two-minute run by applying a voltage of 1000 V (positive-ion detection mode). Ciprofloxacin quantification relied on the ratio of its parent peak (m/z 332) to the IS peak (m/z 372), while identification relied on MS3 of the parent peak and its fragments with m/z values of 314, 288, 268 and 245, respectively. Due to the observed ion suppression effect exerted by the IS over the analyte ion signal at lower concentration levels (< 500 ng/ml ciprofloxacin), the calibration curve was split into two sections. As a result, linearity correlation between the ratios of the analyte to the IS and the corresponding concentrations over the ranges 5–500 and 500–4000 ng/ml respectively was observed. The applicability of the developed method was confirmed by analyzing ciprofloxacin in a pharmaceutical dosage form as well as detection of the analyte in random human clinical urine samples after oral administration of the drug.

Direct nano-electrospray ionization tandem mass spectrometry for the quantification and identification of metronidazole in its dosage form and human urine.

A rapid, sensitive and direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) method, using an offline nanospray (NS) capillary, has been developed and validated for the analysis of metronidazole (MTZ). A mixture of 2 µl MTZ sample solution prepared in an ionization solvent consisting of methanol : water : formic acid in a ratio of 80 : 20 : 0.3, together with 2 µl of an internal standard (IS), 1,3,6-polytyrosine, is loaded into the back of the NS capillary. The NS capillary was fitted into the ion source at a distance of 3 mm between the NS tip and MS orifice. The sample is then analysed and acquired a sustainable signal that allowed for data compilation across various data points for MTZ identification and quantification. The quantification relied on the ratio of the [M + H]+ peaks of MTZ and IS with m/z values of 172.0717 and 182.0812, respectively, while the identification relied on the MS/MS of the precursor ions [M + H]+ of both compounds and their fragments at 128.05 for MTZ and 165.1 and 136.07 for the IS. The NS-ESI-MS/MS method was accurate and precise for the quantification of MTZ over the concentration range from 2.5 to 25 000 ng ml−1. The applicability of the method was confirmed by MTZ analysis in its pharmaceutical dosage form and detection of the analyte in clinical human urine samples without any sample treatment procedure.

Depth-Resolved Nanospray Desorption Electrospray Ionization Mass Spectroscopy in Electrochemically Active Biofilms

Recently, some bacterial biofilms have been shown to colonize and transfer their extracellular electrons to solid electrodes in microbial fuel cells and in bioelectrochemical systems [1, 2]. This class of microorganisms is described as electrochemically active biofilms (EABs) that are able to transfer electrons outside the cell to insoluble electron acceptors (iron or metal oxides) or to solid electrodes via extracellular electron transfer [3, 4]. The need to understand the distribution of proteins involved in electron transfer in EABs is of critical importance. These proteins can be identified using mass spectroscopy (MS). It is known that there is a wide spectrum of applications in which mass spectroscopy (MS) can provide information from a sample of EABs, and these applications can easily translate to all areas of research in EABs. However, the spatial diversity of microbial functions within EABs cannot be resolved because all of the current MS analysis techniques are conducted in the bulk liquid, which provides no spatial resolution with respect to the structure of EABs. Separation techniques can be combined with MS to present a more focused data set with regard to composition; however, MS analysis still lacks the capacity to investigate variation with depth of EABs. Depth profiles will have the potential to elucidate the mechanisms of the surrounding matrix and the roles of the microorganism with respect to the layers of EABs and the growth interface. To date, this MS technique has not been used for depth profiling in EABs. This is mostly because there was no tool available to extract samples from different depths in EABs. Microcapillaries with a several-micrometer tip diameter can be used in EABs without damaging its structure for the depth profiling of selected chemicals. The goal of this work was to develop a microcapillary system which can extract samples at desired depth inside the EABs. Ambient pressure surface ionization mass spectrometry is used to obtain a chemical analyte for sampling from interfaces without special sample preparation [5]. Desorption electrospray ionization (DESI) is an ambient ionization technique in which charged droplets from an electrosonic spray ionization source are aimed towards a surface with a proximal atmospheric pressure mass spectrometer inlet. In this technique, analyte molecules are collected from flat surfaces followed by ionization using a self-aspirating nanoelectrospray. This technique directly transports and ionizes an analyte that is desorbed from a surface into a liquid and it is called as nanospray DESI (nano-DESI). The nanospray capillary transports the charged liquid to the mass spectrometer inlet directly, eliminating splashing while minimizing analyte transport distance. The target application of the developed microcapillary system is to interface it with a nano-DESI sensor which can be used to characterize in situ, depth-resolved analyses of metabolites and possibly proteins. The developed nano-DESI sensor is composed of two microcapillaries placed in an outer case made of glass. Figure 1 shows an image of a developed nano-DESI sensor. In this system, the solvent delivery and its collection were made from the same microcapillaries. We managed to operate the sensor between 10 nL/min and 100 mL/min flow rates. After optimization of the flow rates, we tested it in EABs. EABs were grown according to our previously published paper and book [6, 7]. While we had succeeded in developing a nano-DESI sensor, we found unexpected challenges using it with EABs. Finally, the microcapillary system developed for this work enabled us to use it for quantifying electron transfer processes in EABs [7].

High-resolution mobility analysis of charge-reduced electrosprayed protein ions.

Many mobility studies (IMS) of electrospray ions with charge states z reduced to unity have shown a singular ability to analyze large protein complexes and viruses, though with wide mobility peaks (fwhm ∼ 20%). Here we confirm that this limitation arises primarily when early charge reduction precedes drop evaporation (suppressing secondary atomization by the usual sequence of many Coulomb explosions). By drying before neutralizing, we achieve a protein fwhm of ∼3.7%. A positively biased electrospraying capillary is coaxial with a cylindrical charge-reduction (CR) chamber coated with radioactive Ni-63 (10 mCi) that fills the CR chamber with a bipolar ionic atmosphere. A screen interposed between the spraying capillary and the CR chamber limits penetration of the neutralizing anions into the electrospray (ES) chamber, precluding destabilization of the ES tip, even when brought very close to the grid to enhance ion transmission. As ES cations cross the grid, driven by their own space charge, they recombine with CR ions reducing their charge state as well as space charge dispersion. The setup is tested with the protein ovalbumin (MW ∼ 44.3 kDa) and its clusters up to the tetramer, by analyzing the charge-reduced ions with a differential mobility analyzer (DMA). At gas sample flow rates of ∼1 L/min, the dominant peaks are singly charged (z = 1). They are widened by clustering of involatile solution impurities, depending on spray quality and solution cleanness, with fwhm as small as 3.7% achieved in desalted and acidified solutions. When using sharp nanospray capillaries, the grid may be removed, resulting in ∼2-fold increased ion transmission. In the absence of the grid, however, spray stability and quality are often compromised, even with capillary tip diameters as small as 30 μm.

On‐stage liquid‐phase lipid microextraction coupled to nanospray mass spectrometry for detailed, nano‐scale lipid analysis

Developments in instrumentation aimed at microscopic sampling have led to an emphasis on applications analyzing small volumes and molecular concentrations within biological, chemical, and industrial samples. Simultaneous improvements in the sensitivity and versatility of nanospray mass spectrometers have made it possible to directly couple these sampling and analysis processes. We developed a versatile liquid-phase lipid microextraction (LPME) technique for nanoliter to microliter volumes that is amenable to direct nanospray mass spectrometry (NMS). Lipophilic analytes within several types of biological samples were extracted and analyzed by partitioning and concentrating the analytes based on their solubility within two immiscible or partially miscible liquid phases. The utility of LPME-NMS is demonstrated by extracting and analyzing molecules in four different types of applications: (1) visualization of an extracted neutral lipid-specific fluorescent dye from an aqueous solvent; (2) identification of controlled acid-catalyzed hydrolysis of triacylglycerols within nanospray capillaries; (3) reproducible sampling of a fatty acid emulsion; and (4) profiling of diverse lipids in a complex biological matrix of rabbit serum. The modified instrumentation of a multi-port, on-stage bioworkstation shows considerable versatility by combining nanomanipulation, microextraction and direct NMS for a variety of chemical, biological, industrial, and clinical applications.

MEMS-Based Nanospray-Ionization Mass Spectrometer

An electrospray-ionization mass spectrometer (ESI-MS) whose main components are all fabricated using silicon microelectromechanical systems (MEMS) techniques is demonstrated for the first time. The ion source consists of a microengineered alignment bench containing a V-groove mounting for a nanospray capillary, an ion-extraction electrode, and a pneumatic nebulizer. The vacuum interface consists of two plates, each carrying a 50-μm-diameter capillary, that are selectively etched and bonded together to provide a differentially pumped internal cavity. The quadrupole filter consists of a microfabricated frame that provides mountings for stainless-steel rods measuring 650 μm in diameter and 30 mm in length. Two different quadrupoles are compared: a first-generation bonded silicon device and a second-generation silicon-on-glass device with a Brubaker prefilter. Differential pumping of a MEMS component is demonstrated for the first time, atmospheric pressure ionization and ion transfer into vacuum are characterized, ESI-MS operation is demonstrated, and spectra are presented for a variety of compounds.

Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1(-/-) male mice have normal fertility, Tpst2(-/-) males are infertile despite normal spermatogenesis. However, Tpst2(-/-) sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8(-/-) male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1(-/-), and Tpst2(-/-) epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1(-/-), but not in Tpst2(-/-) mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2(-/-) males.

Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.

Flow-rate characterization of microfabricated polymer microspray emitters.

Microfabricated polymer microspray emitters are characterized in terms of applicable flow rates, temporal and spectral signal-to-noise ratios (SNRs), and solution composition. First, microspray emitters can be operated with 50% methanol/49% water/1% acetic acid from 250 nL/min up to 7 microL/min, with better SNRs above 1 microL/min. Interestingly, even at the lowest flow rates tested, they compare well with nanospray capillaries in terms of mass spectral performances. Secondly, they can be operated with acetonitrile from 10 up to 99% (v/v), with flow rates from 250 nL/min up to 4 microL/min. Even if the mass spectral performances (especially the spectral SNRs) vary with the acetonitrile content, this study validates such microfabricated microspray emitters as interfaces between liquid-phase separations and electrospray mass spectrometers.

Shifts in peptide and protein charge state distributions with varying spray tip orifice diameter in nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry.

The influence of the diameter of the spray tip employed for nano-electrospray mass spectrometry (nano-ES-MS) upon mass spectral charge state distributions was investigated using angiotensin I (M(r) = 1296), insulin (M(r) = 5774), and ubiquitin (M(r) = 8560) as test analytes. Under a variety of experimental conditions, the charge state distributions of the test peptides and protein consistently shifted toward higher values as the tip orifice diameter decreased. This finding indicates that the use of narrow diameter capillaries can promote the formation of higher charge state ions that are more reactive precursors in tandem mass spectrometry experiments. A detailed comparison of charge state distributions obtained for nanospray capillaries of varying diameters was undertaken while systematically varying experimental parameters such as sample flow rate, analyte concentration, solvent composition, and electrospray current. The general tendency to obtain higher charge states from narrow diameter capillaries was conserved throughout, but tips with smaller orifices were more sensitive to sample flow rate (the average charge state was lowered significantly as flow was raised), whereas tips with bigger orifices were more sensitive to analyte concentration and pH of the solution (as each was lowered, the average charge state increased).

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

Subattomole-Sensitivity Microchip Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection

A microfabricated microfluidic device coupled with a nanospray tip for electrospray ionization of dilute solutions is described. The device has been interfaced with a time-of-flight mass spectrometer and evaluated for sensitive, high-speed detection of peptides and proteins. The electrospray voltage was applied through the microchip to the nanospray capillary that was attached at the end of a microfabricated channel. Fluid delivery rates were 20-30 nL min(-)(1) through the hybridized structure without any pressure assistance. On-line microchip electrophoresis has been demonstrated and the effect of the capillary-chip junction on band broadening examined. Full mass spectra are acquired within 10-20 ms at 50-100 spectra s(-)(1) storage rates. Detection of subattomole levels of sample from a 100 nM solution is demonstrated for infusion experiments.

Evaluation of heat-induced conformational changes in proteins by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry

The characterization of heat-induced conformational changes of proteins by nanoelectrospray mass spectrometry is shown. The used device is the first to make a simple and effective investigation of the denaturation of proteins possible using minimum sample consumption, as enabled by nanoelectrospray mass spectrometry. As indicated by average charge states at several solution temperatures, a thermal induced denaturation of ubiquitin, in the temperature range between 25 and 90°C, was investigated. A single structure transition of ubiquitin was observed. Heating and unassisted cooling by air convection of the sample solution occurs directly inside the nanospray capillary during the electrospray measurement. Therefore, this new device is also suitable for probing for the reversibility of conformational changes in proteins.