Nanospray Tip Research Articles

Discovery of Potential Lipid Biomarkers for Human Colorectal Cancer by In-Capillary Extraction Nanoelectrospray Ionization Mass Spectrometry.

Discovering cancer biomarkers is of significance for clinical medicine and disease diagnosis. In this article, we develop an in-capillary extraction nanoelectrospray ionization mass spectrometry (ICE-nanoESI-MS) method to rapidly and in situ investigate human colorectal cancer for discovering lipid biomarkers. The ICE-nanoESI-MS method is performed using a tungsten microdissecting probe for in situ microsampling of surgical human colorectal cancer tumors and their paired distal noncancerous tissues during/after surgery. After sampling, the tungsten probe and the adhered tissues are inserted into a nanospray tip prefilled with some solvent for simultaneous in-capillary extraction and nanoESI-MS detection under ambient and open-air conditions. Online coupling of the Paternò-Büchi reaction and radical-direct fragmentation with ICE-nanoESI-MS is easily realized, which provides the opportunity to precisely determine carbon-carbon double bond (C═C) locations and stereospecific numbering (sn) positions of lipid biomarkers. Subsequently, a total of 12 pairs of colorectal cancer tumors and distal noncancerous tissues from different patients are investigated by our proposed ICE-nanoESI-MS method. A significant increase in lysophospholipids and fatty acids as well as a significant decrease in ceramides are discovered, and lysophospholipids are found as the potential biomarkers related to the formation and pathogenesis of human colorectal cancer.

Analysis of the intracellular localization of amiodarone using live single-cell mass spectrometry

Amiodarone is a well-known antiarrhythmic drug with side effects including phospholipidosis. However, it is not clear how amiodarone and its metabolites are localized in the cell. In the present study, the localization of amiodarone in the cytosol, vacuoles, and lipid droplets of a single HepG2 human hepatocellular carcinoma cell was determined directly using live single-cell mass spectrometry. The cytosol, vacuoles, and lipid droplets of a single HepG2 cell treated with amiodarone were separately captured using a nano-spray tip under a fluorescence microscope after visualizing the lipid droplets using a fluorescent probe. This assay showed a linearity in the measurement of amiodarone levels with R2 values of 0.9996 and 0.9998 in the cell lysates and serum, respectively. The peak intensities of amiodarone and its metabolites in lipid droplets and vacuoles were significantly higher than those in the cytosol, while those in lipid droplets were higher than those in vacuoles. Amiodarone metabolites were detected in both lipid droplets and the cytosol. Live single-cell mass spectrometry combined with fluorescence imaging demonstrated clear localization of amiodarone and its metabolites in lipid droplets separately from the vacuole. This assay system combined with fluorescence imaging could be useful for investigating the intracellular localization of various drugs and their metabolites.

Analysis of Live Single Cells by Confocal Microscopy and High-Resolution Mass Spectrometry to Study Drug Uptake, Metabolism, and Drug-Induced Phospholipidosis.

The analysis of large numbers of cells from a population results in information that does not reflect differences in cell phenotypes. Individual variations in cellular drug uptake, metabolism, and response to drug treatment may have profound effects on cellular survival and lead to the development of certain disease states, drug persistence, and resistance. Herein, we present a method that combines live cell confocal microscopy imaging with high-resolution mass spectrometry to achieve absolute cell quantification of the drug amiodarone (AMIO) and its major metabolite, N-desethylamiodarone (NDEA), in single liver cells (HepG2 and HepaRG cells). The method uses a prototype system that integrates a confocal microscope with an XYZ stage robot to image and automatically sample selected cells from a sample compartment, which is kept under growth conditions, with nanospray tips. Besides obtaining the distributions of AMIO and NDEA cell concentrations across a population of individual cells, as well as variabilities in drug metabolism, the effect of these on phospholipidosis and cell morphology was studied. The method was suited to identify subpopulations of cells that metabolized less drug and to correlate cell drug concentrations with cell phospholipid content, cell volume, sphericity, and other cell phenotypic features. Using principal component analysis (PCA), the treated cells could be clearly distinguished from vehicle control cells (0 μM AMIO) and HepaRG cells from HepG2 cells. The potential of using multidimensional and multimodal information collected from single cells to build predictive models for cell classification is demonstrated.

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.

Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C═C Location Isomers in Complex Biological Tissues.

Lipids are important structural components of biological systems, and lipid C═C locations play important roles in their biophysical and biochemical properties. Rapid, in vivo, in situ, and microscale lipidomics investigation (including precise identification of lipid C═C locations and isomers) of biological specimen has great potential for clinical diagnosis, biological studies, and biomarker discovery. Here we report a novel lipidomics methodology by coupling Paternò-Büchi (PB) reaction with surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) for in vivo, in situ, and microscale analysis of lipid species and C═C location isomers in complex biological tissues. The proposed SCP-PB-nanoESI-MS method was performed by application of a biocompatible solid-phase microextraction (SPME) probe for in vivo, in situ, and microscale sampling and extraction of lipids from biological tissues, and then some spray solvent containing PB reagent was applied to desorb lipid species enriched on SPME probe within a nanospray tip. Subsequently, ultraviolet irradiation was employed to initiate PB reaction for unsaturated lipids within the nanospray tip. After that, a high voltage was applied on the SPME probe to induce nanoESI for MS analysis under ambient and open-air conditions, and collision-induced dissociation was performed to the PB reaction product ions for determination of lipid C═C locations and isomers. By using our proposed SCP-BP-nanoESI-MS method, microscale investigation of lipid compositions and C═C location isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C═C locations for zebrafish was accomplished.

Quantitative Live Single-cell Mass Spectrometry with Spatial Evaluation by Three-Dimensional Holographic and Tomographic Laser Microscopy

The locations and volumes of the contents of a single HepG2 cell were visualized under three-dimensional (3D) holographic and tomographic (HT) laser microscopy, colored by refractive index, not staining. After trapping the specific area of a target cell in a nanospray tip, quantification was performed by live single-cell mass spectrometry. Comparison of the HepG2 cells' before and after 3D-HT images allowed the inference of the precise volume and original location of the trapped cell contents. The total amount of a trapped molecule was estimated. The images also revealed morphological changes in the cell structure caused by the manipulation.

Lateral resolution of desorption nanoelectrospray: a nanospray tip without nebulizing gas as a source of primary charged droplets.

Desorption nanoelectrospray (nanoDESI) was described in 2007 and it represents a miniaturized version of desorption electrospray without the assistance of the nebulizing gas. Compared to DESI, a nanoelectrospray tip (2 ± 1 μm I.D.) generates primary charged droplets of smaller sizes and lower spray liquid flow rates. This is the first report on utilization of nanoDESI for mass spectrometry imaging (MSI). Its new coupling with a Q-TOF instrument allowed faster mass spectra acquisition (4 Hz) essential for MSI of fine surface details. To evaluate nanoDESI potential for mass spectrometry imaging, etched glass substrates with Rhodamine B patterns of different dimensions were prepared. The Rhodamine B lines were analysed in 1D scanning mode and their width was determined experimentally by nanoDESI measurement. The experimental data revealed that the lateral resolution of nanoDESI is close to 30 μm along the x-axis (orthogonal to the inlet). 2D scanning mode confirmed good resolution along both axes as dye squares with dimensions about 60 μm × 60 μm were easily distinguished. The low flow rate of the spray liquid reduced undesirable analyte washing effects, which allowed repeated scanning analysis of the surface. The presented results demonstrate the applicability of nanoDESI for high surface resolution mass spectrometry imaging.

Reproducible preparation of nanospray tips for capillary electrophoresis coupled to mass spectrometry using 3D printed grinding device.

The use of high quality fused silica capillary nanospray tips is critical for obtaining reliable and reproducible electrospray/MS data; however, reproducible laboratory preparation of such tips is a challenging task. In this work, we report on the design and construction of low-cost grinding device assembled from 3D printed and commercially easily available components. Detailed description and characterization of the grinding device is complemented by freely accessible files in stl and skp format allowing easy laboratory replication of the device. The process of sharpening is aimed at achieving maximal symmetricity, surface smoothness and repeatability of the conus shape. Moreover, the presented grinding device brings possibility to fabricate the nanospray tips of desired dimensions regardless of the commercial availability. On several samples of biological nature (reserpine, rabbit plasma, and the mixture of three aminoacids), performance of fabricated tips is shown on CE coupled to MS analysis. The special interest is paid to the effect of tip sharpness.

Direct Lipido-Metabolomics of Single Floating Cells for Analysis of Circulating Tumor Cells by Live Single-cell Mass Spectrometry.

Direct trapping of a single floating cell, i.e. a white blood cell from a drop of blood, within a nanospray tip was followed by super-sonication after the addition of ionization solvent. Molecular detection of an increased number of peaks with a higher intensity and a wider m/z range, which extends from metabolites to lipids, was acquired than of that without sonication. This method was applied to a few separated circulating tumor cells (CTC) from a neuroblastoma patient's blood to obtain their lipido-metabolomic molecular profile at the single cell level. In addition to vital molecules such as amino acids, catechol amine metabolites, which are specific to neuroblastoma, and drugs included in the patient's course of therapy were detected. This established "direct single-cell lipido-metabolomic method" seems to be useful for direct and wide range molecular detection not only for many live single-cells, but also for rare cells, such as CTCs, for future molecular diagnosis.

Fluorescence Probing Live Single-cell Mass Spectrometry for Direct Analysis of Organelle Metabolism.

Mitochondria in a live HepG2 cell were visualized with a fluorescent probe to specify their location and state in a living cell. Then, mitochondria were selectively captured with a nanospray tip under fluorescence microscope, and thousands of small molecular peaks were revealed and unique steroids specific to mitochondria were also found. This fluorescence imaging combined with live single-cell mass spectrometry opens the door to the analysis of site- and state-specific molecular detection to elucidate precise molecular mechanisms at the single-cell and organelle level.

Mechanism of C-Terminal Fragments of Amyloid β-Protein as Aβ Inhibitors: Do C-Terminal Interactions Play a Key Role in Their Inhibitory Activity?

Targeting the early oligomerization of amyloid β protein (Aβ) is a promising therapeutic strategy for Alzheimer's disease (AD). Recently, certain C-terminal fragments (CTFs) derived from Aβ42 were shown to be potent inhibitors of Aβ-induced toxicity. The shortest peptide studied, Aβ(39-42), has been shown to modulate Aβ oligomerization and inhibit Aβ toxicity. Understanding the mechanism of these CTFs, especially Aβ(39-42), is of significance for future therapeutic development of AD and peptidomimetic-based drug development. Here we used ion mobility spectrometry-mass spectrometry to investigate the interactions between two modified Aβ(39-42) derivatives, VVIA-NH2 and Ac-VVIA, and full-length Aβ42. VVIA-NH2 was previously shown to inhibit Aβ toxicity, whereas Ac-VVIA did not. Our mass spectrometry analysis revealed that VVIA-NH2 binds directly to Aβ42 monomer and small oligomers while Ac-VVIA binds only to Aβ42 monomer. Ion mobility studies showed that VVIA-NH2 modulates Aβ42 oligomerization by not only inhibiting the dodecamer formation but also disaggregating preformed Aβ42 dodecamer. Ac-VVIA also inhibits and removes preformed Aβ42 dodecamer. However, the Aβ42 sample with the addition of Ac-VVIA clogged the nanospray tip easily, indicating that larger aggregates are formed in the solution in the presence of Ac-VVIA. Molecular dynamics simulations suggested that VVIA-NH2 binds specifically to the C-terminal region of Aβ42 while Ac-VVIA binds dispersedly to multiple regions of Aβ42. This work implies that C-terminal interactions and binding to Aβ oligomers are important for C-terminal fragment inhibitors.

Capillary electrophoresis in an extended nanospray tip–electrospray as an electrophoretic column

Capillary electrophoresis coupled to mass spectrometry (CE/MS) is gaining its space among the most powerful tools in modern (bio)analytical laboratory. The most challenging instrumental aspect in CE/MS is striking the balance between the stability and reproducibility of the signal and sensitivity of the analysis. Several interface designs have been published in the past decade addressing the variety of instrumental aspects and ease of operation. Most of the interfaces can be categorized either into the sheath flow arrangement (considered to be a de facto standard), or sheathless interface, often expected to provide the ultimate sensitivity. In this work we have explored an “interface-free” approach, where the CE/MS analysis was performed in narrow bore (<20μm ID) electrospray capillary. The separation capillary and electrospray tip formed one entity and the high voltage, applied at the injection end of the capillary served for both the separation and electrospray ionization. Thus the separation voltage was defined as the product of the electrospray current and resistivity of the separation electrolyte. Optimum conditions for the separation and electrospray ionization were achieved with voltage programming. The performance of this simplest possible CE/MS system was tested on peptide separations from the cytochrome c tryptic digest. The subnanoliter sample consumption and sensitivity in the attomole range predetermines such a system for analysis of limited samples.

Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.

Direct Drug Metabolism Monitoring in a Live Single Hepatic Cell by Video Mass Spectrometry

The metabolism of anti-breast cancer drug, tamoxifen, in a single human hepatocellular carcinoma cell, HepG2, was directly monitored by a video-mass spectroscope. The cytoplasm, a vacuole or nucleus of the cell was directly sucked by a nano-spray tip under a video-microscope, and then was introduced into a mass spectrometer. Unchanged drug molecules were found in cytoplasm and a vacuole, but the metabolites were only found in the cytoplasm. This direct detection of drug metabolites in a live single cell is useful for speedy drug metabolism monitoring.

Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair

A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.

Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation

In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795–806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.

Nanomanipulation‐Coupled Nanospray Mass Spectrometry Applied to the Extraction and Analysis of Trace Analytes Found on Fibers*

This article presents the novel instrumentation of nanomanipulation coupled to nanospray ionization-mass spectrometry, which is used to directly probe trace analytes found on individual fibers. The low detection limits and sample volumes associated with nanospray ionization-mass spectrometry make it the ideal instrument to implement for trace analysis. Nanospray ionization-mass spectrometry, coupled with the nanomanipulator, allows for the direct probing of trace particulates on fibers. The technique is demonstrated by dissolving an electrostatic particle of cocaine from a fiber, collecting the analyte solution in a nanospray tip, and transferring the tip directly to the mass spectrometer to complete analysis. The utility of this technique is evident through the minimal sample preparation and short analysis time. The use of nanomanipulation coupled to nanospray ionization-mass spectrometry could improve on current trace particulate analysis by reducing both detection limits and sample size required to complete analysis.

Focus on spectroscopy

Focus on spectroscopy

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.