RESUMEN
Discriminating various leukocyte subsets with specific functions is critical due to their important roles in the development of many diseases. Here, we proposed a general strategy to unravel leukocytes heterogeneity and screen differentiated metabolites as biomarker candidates for leukocyte subtypes using the label-free mass cytometry (CyESI-MS) combined with a homemade data processing workflow. Taking leukemia cells as an example, metabolic fingerprints of single leukemia cells were obtained from 472 HL-60, 416 THP-1, 313 U937, 356 Jurkat, and 366 Ramos cells, with throughput up to 40 cells/min. Five leukemia subtypes were clearly distinguished by unsupervised learning t-SNE analysis of the single-cell metabolic fingerprints. Cell discrimination in the mixed leukemia cell samples was also realized by supervised learning of the single-cell metabolic fingerprints with high recovery and good repetition (98.31 ± 0.24%, -102.35 ± 4.82%). Statistical analysis and metabolite assignment were carried out to screen characteristic metabolites for discrimination and 36 metabolites with significant differences were annotated. Then, differentiated metabolites for pairwise discrimination of five leukemia subtypes were further selected as biomarker candidates. Furthermore, discriminating cultured leukemia cells from human normal leukocytes, separated from fresh human peripheral blood, was performed based on single-cell metabolic fingerprints as well as the proposed biomarker candidates, unveiling the potential of this strategy in clinical research. This work makes efforts to realize high-throughput single-leukocyte metabolic analysis and metabolite-based discrimination of leukocytes. It is expected to be a powerful means for the clinical molecular diagnosis of hematological diseases.
Asunto(s)
Leucemia , Biomarcadores , Humanos , Leucemia/diagnóstico , LeucocitosRESUMEN
Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10-18 mol) to fmol (10-15 mol), providing the foundation of metabolite quantitation of single cells. We applied this method to investigate the changes of metabolites including glucose-phosphate, 2-deoxy-d-glucose-phosphate, and ribose-phosphate in single K562 cells stimulated by 2-deoxy-d-glucose. With the robust quantitative capabilities, the developed method holds great potential for studying a drugs' mechanism of action and resistance at single cell level.
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Glucofosfatos/análisis , Glucofosfatos/aislamiento & purificación , Dispositivos Laboratorio en un Chip , Microextracción en Fase Líquida/instrumentación , Espectrometría de Masas/instrumentación , Análisis de la Célula Individual/métodos , Humanos , Células K562RESUMEN
We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.
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Cebollas/química , Compuestos Orgánicos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Células HeLa , Humanos , Metabolómica/economía , Metabolómica/métodos , Cebollas/metabolismo , Compuestos Orgánicos/economía , Compuestos Orgánicos/metabolismo , Análisis de la Célula Individual/economía , Análisis de la Célula Individual/métodos , Espectrometría de Masa por Ionización de Electrospray/economíaRESUMEN
In this study, we developed a probe-electrospray ionization method by coupling a SPME probe modified with nanosized TiO2 directly to nanoESI-MS for the phosphoproteome analysis, which demonstrated excellent selectivity and sensitivity for enrichment of phosphopeptides in complex biological samples.
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Fosfopéptidos/análisis , Fosfopéptidos/aislamiento & purificación , Proteómica/métodos , Microextracción en Fase Sólida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Humanos , Fosfopéptidos/sangre , Fosfopéptidos/química , Propiedades de Superficie , Titanio/químicaRESUMEN
Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 µm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell "omics".
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Células/química , Metabolómica/métodos , Análisis de la Célula Individual/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Fracciones Subcelulares/química , Angiotensina II/análisis , Fructanos/análisis , Cebollas/química , Cebollas/citología , Raíces de Plantas/química , Compuestos de Tungsteno/químicaRESUMEN
Matrix unloaded: By changing from fixed-voltageâ (left) to step-voltage nanoelectrosprayâ (right), the mass-spectrometric analysis of small-volume physiological samples is possible. Separation and ionization are achieved in one process, which avoids sample loss and dilution and prevents interference by the matrix. The result is high sensitivity even for samples at the nanoliter level.
RESUMEN
The method for the localization of bioactive molecules in plants is highly needed since it provides a fundamental prerequisite for understanding their physiological and ecological functions. Here, we propose a simple method termed in vivo nanoelectrospray for the localization of bioactive molecules in plants without sample preparation. A capillary is partly inserted into the plant to sample liquid from a highly located region, and then, a high voltage is applied to the plant to generate an electrospray from the capillary tip for mass spectrometry analysis. Using this method, bioactive molecules such as saccharides, glycoalkaloids, flavonoids, organic acids, and glucosinolates (GLs) are detected in the target regions of living plants or fresh fruits. Original information for endogenous chemicals including liable molecules in plant can be obtained. A sketchy three-dimensional distribution of glycoalkaloids in a cherry tomato has been obtained. The present work provides a powerful tool for the study of bioactive molecules in a living plant by mass spectrometry.
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Productos Biológicos/análisis , Nanotecnología/métodos , Plantas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Productos Biológicos/química , Plantas/metabolismoRESUMEN
A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions.
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Péptidos/química , Espectrometría de Masa por Ionización de Electrospray , Dinorfinas/química , Electroforesis Capilar , Encefalina Metionina/análogos & derivados , Encefalina Metionina/química , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Timosina/químicaRESUMEN
A new low-temperature plasma (LTP), based on dielectric barrier discharge (DBD), has been developed as an alternative ionization source for ambient mass spectrometry. For organic samples, the source is able to produce two different fragmentation patterns which are selectable by an electrical switch. The two source modes are different only in the second electrodes: in configuration (A), bar-plate and in configuration (B), coaxial bar-cylinder shapes are used. A disposable graphite probe is used as the first electrode, the same in both configurations, and a copper foil is used as the second electrode. The ionization source is applicable to gas and liquid samples, without any change being necessary in its design. Under optimal conditions, to take ethylbenzene as an example, a detection limit of less than 25 ng was obtained and a relative standard deviation (RSD) of 13.36% has been demonstrated for 50 ng of ethylbenzene (n = 11). We have found several interesting differences in the mass spectra of the tested volatile organic compounds (VOCs) in the two modes, which might be applicable in identification studies. We have investigated the effect of variation of the first electrode material and the second electrode length in mode B. Moreover, in this design the graphite electrode is capable of acting as a sample adsorbent, which is a new sampling method for LTP mass spectrometry (MS). This capability was investigated by adsorption of the selected VOCs onto the surface of the graphite electrode in a headspace solid-phase microextraction (SPME) system, and direct desorption and ionization of the samples by LTPMS.
RESUMEN
A simple and fast (<5 s) method for in situ arsenic speciation on solid surfaces has been developed based on desorption electrospray ionization-tandem mass spectrometry (DESI-MS). Arsenic-polluted environmental samples such as animal feed and plant tissues could be directly monitored by DESI-MS. Each arsenic species in this study, including monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB), arsenocholine (AsC), 4-arsanilic acid (p-ASA), 4-hydroxyphenylarsonic acid (4-OH), Nitarsone, Roxarsone and two inorganic arsenic species, arsenate As(v) and arsenite As(iii), could be detected by their typical m/z and collision induced dissociation (CID) behavior respectively. By the characteristic information, mixtures of different arsenic species could be detected without any sample preparation and separation process. This method could give absolute detection limits of the arsenic species at ng/mm(2) to pg/mm(2) level with a best RSD of 5.3% (n = 5). The method could be potentially applied to in situ environmental monitoring of arsenic pollution, especially that caused by arsenic pesticides, animal feed additives, herbicides and wood treatment.
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Arsénico/análisis , Contaminantes Ambientales/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Alimentación Animal/análisis , Monitoreo del Ambiente , Plaguicidas/química , Espectrometría de Masas en TándemRESUMEN
Occurrence and fate of eight kinds of selected endocrine-disrupting compounds (EDCs) in three sewage treatment plants (STPs) of Beijing, China was investigated. These EDCs, composed of 4-octylphenol (4-OP), 4-n-nonylphenol (4-n-NP), bisphenol A (BPA), estrone (E1), 17alpha-estradiol (17alpha-E2), 17beta-estradiol (E2), estriol (E3) and 17alpha-ethinylestradiol (EE2), in every step of STPs, were simultaneously analysed by gas chromatography/mass spectrometry after derivatisation. All the EDCs were detected in the influents of three STPs, and BPA was the most abundant compound. The concentrations of EDCs ranged from 36.6 ng/l of 17alpha-E2 (STP C) to 1342.3 ng/l of BPA (STP B) in the influent sewages and from below limits of detection of E2 and E3 (STP C) to 142.5 ng/l of E1 (STP B) in the effluent sewages. The STPs could not remove alkylphenols effectively from the aqueous phase with less than 40% reduction. BPA decreased over 90%, and steroid estrogens achieved considerable reductions from 64.8% of E2 to 94.9% of E3. Generally, biological treatment was more effective in removing alkylphenols, BPA and natural estrogens from the aqueous phase than primary treatment. However, the synthetic estrogen, EE2, was mostly removed by the primary treatment with about 63.5% reduction. It is the first time that the concentration of 17alpha-E2 in the sewage of China was reported in this paper. The compound might have a bearing with the waste effluents of dairy farms around urban area of Beijing.
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Disruptores Endocrinos/análisis , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/química , Compuestos de Bencidrilo , China , Disruptores Endocrinos/química , Estradiol/análisis , Estradiol/química , Estrona/análisis , Estrona/química , Estructura Molecular , Fenoles/análisis , Fenoles/química , Contaminantes Químicos del Agua/análisisRESUMEN
Real-time and in-situ monitoring of ongoing chemical reactions by mass spectrometry was achieved by simply directing the low-temperature plasma (LTP) to the surface of the reaction system for analyte desorption and ionization without any sample pretreatment.
RESUMEN
In this paper, we have constructed a low temperature plasma (LTP) probe using dielectric barrier discharge (DBD) and employed it for the detection of explosives on a variety of substrates under ambient conditions. Upon discharge, a transient, low-temperature non-equilibrium plasma comprising ions, electrons and metastable atoms are generated between the electrodes. Three common explosives, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-trinitro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN), were directly desorbed and ionized from solid surfaces, followed by subsequent analysis using the mass spectrometer in the negative ion mode. Limits of detection (LODs) were 500 fg for TNT, 1 pg for RDX, and 500 fg for PETN. The reliability of the method was characterized by a successful analysis of a mixture of the three explosives. The ion source also allowed direct detection of trace explosives on both conductive and non-conductive substrates, thus expanding the applicability of low temperature plasma desorption mass spectrometry.
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Contaminantes Ambientales/análisis , Sustancias Explosivas/análisis , Espectrometría de Masas/métodos , Electroquímica/métodos , Tetranitrato de Pentaeritritol/análisis , Sensibilidad y Especificidad , Temperatura , Triazinas/análisis , Trinitrotolueno/análisisRESUMEN
A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry.
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Espectrometría de Masas/instrumentación , Preparaciones Farmacéuticas/análisis , Frío , Diseño de Equipo , Espectrometría de Masas/economía , Espectrometría de Masas/métodos , Estructura Molecular , Factores de TiempoRESUMEN
Lipids exert substantial influences on vertebrate embryogenesis, but their metabolic dynamics at detailed structural levels remains elusive, primarily owing to the lack of a tool capable of resolving their huge structural diversity. Herein, we present the first large-scale and spatiotemporal monitoring of unsaturated lipids with C=C specificity in single developing zebrafish embryos enabled by photochemical derivatization and tandem mass spectrometry (MS). The lipid isomer composition was found extremely stable in yolk throughout embryogenesis, while notable differences in ratios of C=C location (e.g., PC 16:0_16:1 (7) vs. 16:0_16:1 (9)) and fatty acyl composition isomers (e.g., PC 16:1_18:1 vs. 16:0_18:2) were unveiled between blastomeres and yolk from zygote to 4 h post fertilization (hpf). From 24 hpf onwards, lipid isomer compositions in embryo head and tail evolved distinctively with development, suggesting a meticulously regulated lipid remodeling essential for cell division and differentiation. This work has laid the foundation for functional studies of structurally defined lipids in vertebrate embryology.
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Metabolismo de los Lípidos , Lípidos/análisis , Pez Cebra/embriología , Animales , Ácidos Grasos Insaturados/análisis , Ácidos Grasos Insaturados/metabolismo , Isomerismo , Espectrometría de Masas/métodos , Pez Cebra/metabolismoRESUMEN
A simple and easy-to-build high-throughput analysis system was constructed. The system consisted of three major components: (1) a multichannel device with 16 parallel capillaries, (2) a desorption electrospray ionization (DESI) source, and (3) a linear ion trap mass spectrometer. When analyses were performed, the multichannel device was moved horizontally on a translation stage controlled by a step motor. Our design expands the functions of DESI, in which the liquid sample in capillary was driven out by the nebulizing gas, ionized, and then transferred to a mass spectrometer. To assess the high-throughput performance of the system, 5 mg/L 1,3-diethyl-1,3-diphenylurea (DDU) solution and 10 mg/L angiotensin I solution were alternatively loaded into the reservoirs and capillaries in the multichannel device. Results indicated that analyses of the all the samples in 16 capillaries were completed within 1.6 min, which means a throughput of 600 samples/h. Reactive DESI experiment was also successfully performed with this system to show the feasibility of online derivatization. The relative standard deviations for a single capillary and five identical capillaries were 7.6 (n = 16) and 12.3%, respectively. Linear relative abundance response was achieved for DDU (r = 0.9971).
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Espectrometría de Masa por Ionización de Electrospray/instrumentación , Angiotensina I/análisis , Carbanilidas/análisis , Diseño de Equipo , Estudios de Factibilidad , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
To synthesize aristolochic acid (AA)-2'-deoxyguanosine 5'-monophosphate (dGp) adducts in vitro and develop a novel method for the characterization of the adducts using multiple mass spectrometric techniques. AA was incubated with dGp in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-dGp adducts, and the reaction conditions were optimized. Crude extracts were analyzed by techniques of liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-MS/MS) and high accuracy mass data and isotope pattern of super high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS). The quasi-molecular ion peaks of the AA-dGp adducts were obtained in the negative ion mode. Analysis by electrospray ionization/tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-dGp adducts. AA can bind covalently to the exocyclic amino group of deoxyguanosine to form AA-dGp adducts. MS analysis is a powerful tool to detect and identify AA-dGp adducts simply, rapidly and accurately.
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Ácidos Aristolóquicos/síntesis química , Aductos de ADN/síntesis química , ADN/química , Desoxiguanosina/química , Ácidos Aristolóquicos/química , Cromatografía Líquida de Alta Presión/métodos , ADN/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
A new ion source based on dielectric barrier discharge was developed as an alternative ionization source for ambient mass spectrometry. The dielectric barrier discharge ionization source, termed as DBDI herein, was composed of a copper sheet electrode, a discharge electrode, and a piece of glass slide in between as dielectric barrier as well as sample plate. Stable low-temperature plasma was formed between the tip of the discharge electrode and the surface of glass slide when an alternating voltage was applied between the electrodes. Analytes deposited on the surface of the glass slide were desorbed and ionized by the plasma and the ions were introduced to the mass spectrometer for mass analysis. The capability of this new ambient ion source was demonstrated with the analysis of 20 amino acids, which were deposited on the glass slide separately. Protonated molecular ions of [M + H](+) were observed for all the amino acids except for L-arginine. This ion source was also used for a rapid discrimination of L-valine, L-proline, L-serine and L-alanine from their mixture. The limit of detection was 3.5 pmol for L-alanine using single-ion-monitoring (SIM). Relative standard deviation (RSD) was 5.78% for 17.5 nmol of L-alanine (n = 5). With the advantages of small size, simple configuration and ease operation at ambient conditions, the dielectric barrier discharge ion source would potentially be coupled to portable mass spectrometers.
RESUMEN
Trace amounts of explosives on solid surfaces were detected by mass spectrometry at ambient conditions with a new technique termed dielectric barrier discharge ionization (DBDI). By the needle-plate discharge mode, a plasma discharge with energetic electrons was generated, which could launch the desorption and ionization of the explosives from solid surfaces. Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) were desorbed directly from the explosives-contaminated surface by DBDI, forming the typical anions of [TNT](-), [TNT - H](-), [RDX + NO(2)](-), [PETN + ONO(2)](-), and [RDX + ONO(2)](-). The ions were transferred into the MS instrument for analysis in the negative ion mode. The detection limit of present method was 10 pg for TNT (m/z 197, S/N 8 : 1), 0.1 ng for RDX (m/z 284, S/N 10 : 1), and 1 ng for PETN (m/z 260, S/N 12 : 1). The present method allowed the detection of trace explosives on various matrices, including paper, cloth, chemical fiber, glass, paints, and soil. A relative standard deviation of 5.57% was achieved by depositing 100 pg of TNT on these matrices. The analysis of A-5, a mixture of RDX and additives, has been carried out and the results were consistent with the reference values. The DBDI-MS method represents a simple and rapid way for the detection of explosives with high sensitivity and specificity, which is especially useful when they are present in trace amounts on ordinary environmental surfaces.
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Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.