116 research outputs found

    Analytical techniques for single-cell metabolomics: state of the art and trends

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    Single-cell metabolomics is an emerging field that addresses fundamental biological questions and allows one to observe metabolic phenomena in heterogeneous populations of single cells. In this review, we assess the suitability of different detection techniques and present considerations on sample preparation for single-cell metabolomics. Although targeted analysis of single cells can readily be conducted using fluorescent probes and optical instruments (microscopes, fluorescence detectors), a comprehensive metabolomic approach requires a powerful label-free method, such as mass spectrometry (MS). Mass-spectrometric techniques applied to study small molecules in single cells include electrospray MS, matrix-assisted laser desorption/ionization MS, and secondary ion MS. Sample preparation is an important aspect to be taken into account during further development of methods for single-cell metabolomic

    Negative mode nanostructure-initiator mass spectrometry for detection of phosphorylated metabolites

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    The chemical complexity of the metabolome requires the development of new detection methods to enlarge the range of compounds detectable in a biological sample. Recently, a novel matrix-free laser desorption/ionization method called nanostructure-initiator mass spectrometry (NIMS) [Northen et al., Nature 449(7165):1033-1036, 2007] was reported. Here we investigate NIMS in negative ion mode for the detection of endogenous metabolites, namely small phosphorylated molecules. 3-Aminopropyldimethylethoxysilane was found to be suitable as initiator for the analytes studied and a limit of detection in the tens of femtomoles was reached. The detection of different endogenous cell metabolites in a yeast cell extract is demonstrate

    Interfacing Microfluidics and Laser Desorption/Ionization Mass Spectrometry by Continuous Deposition for Application in Single Cell Analysis

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    We present a simple method for continuous deposition of effluent originating from a microfluidic device on a flat metal surface for subsequent analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The sample is delivered using a microscale fused silica capillary and passed onto the surface of a stainless steel plate coated with a layer of a standard matrix. The key parameters optimized in order to obtain high quality and reproducible sample traces are: i) sampleflow rate, ii) speed of the XY-stage movement, and iii) distance of the capillary tip from the plate. Tapering the capillary end as well as surface functionalization to induce hydrophobicity were shown to further enhance the deposition process. The described continuous deposition method is compared with a previously published mass spectrometric method utilizing a piezoelectric microdispenser for microspotting onto the MALDI plates which enabled detection of primary metabolites at the singlecell level. Research is underway to adapt the continuous deposition as an interface for single cell metabolite detection and enhancement of quantitative abilities of the MALDI methodology. We envisage that the presented continuous deposition method may also be suitable for sensitive detection of analytes using other surface analysis tools

    MALDI-MS analysis and imaging of smallmolecule metabolites with 1,5-diaminonaphthalene (DAN)

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    1,5-diaminonaphthalene (DAN) has previously been reported as an effective matrix for matrix-assisted laser desorption ioni- zation-mass spectrometry of phospholipids. In the current work, we investigate the use of DAN as a matrix for small metab- olite analysis in negative ion mode. DAN was found to provide superior ionization to the compared matrices for MW \u3c ~400 Da; however, 9-aminoacridine (9-AA) was found to be superior for a uridine diphosphate standard (MW 566 Da). DAN was also found to provide a more representative profile of a natural phospholipid mixture than 9-AA. Finally, DAN and 9-AA were applied for imaging of metabolites directly from corn leaf sections. Published 2014. This article is a U.S. Government work and is in the public domain in the USA

    QSpec: online control and data analysis system for single-cell Raman spectroscopy

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    Single-cell phenotyping is critical to the success of biological reductionism. Raman-activated cell sorting (RACS) has shown promise in resolving the dynamics of living cells at the individual level and to uncover population heterogeneities in comparison to established approaches such as fluorescence-activated cell sorting (FACS). Given that the number of single-cells would be massive in any experiment, the power of Raman profiling technique for single-cell analysis would be fully utilized only when coupled with a high-throughput and intelligent process control and data analysis system. In this work, we established QSpec, an automatic system that supports high-throughput Raman-based single-cell phenotyping. Additionally, a single-cell Raman profile database has been established upon which data-mining could be applied to discover the heterogeneity among single-cells under different conditions. To test the effectiveness of this control and data analysis system, a sub-system was also developed to simulate the phenotypes of single-cells as well as the device features

    Natural Occurrence of 2′,5′-Linked Heteronucleotides in Marine Sponges

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    2′,5′-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2′,5′-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2′-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2′,5′-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2′,5′-linked heteronucleotides which accompanied high levels of 2′,5′-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2′p5′G, A2′ p5′U, A2′p5′C, G2′p5′A and G2′ p5′U. The natural occurrence of 2′-adenylated NAD+ was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2′,5′-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family

    Microfluidic Technologies for Synthetic Biology

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    Microfluidic technologies have shown powerful abilities for reducing cost, time, and labor, and at the same time, for increasing accuracy, throughput, and performance in the analysis of biological and biochemical samples compared with the conventional, macroscale instruments. Synthetic biology is an emerging field of biology and has drawn much attraction due to its potential to create novel, functional biological parts and systems for special purposes. Since it is believed that the development of synthetic biology can be accelerated through the use of microfluidic technology, in this review work we focus our discussion on the latest microfluidic technologies that can provide unprecedented means in synthetic biology for dynamic profiling of gene expression/regulation with high resolution, highly sensitive on-chip and off-chip detection of metabolites, and whole-cell analysis

    Analytical techniques for single-cell metabolomics: State of the art and trends

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    ISSN:1618-2650ISSN:1618-264
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