Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

Amantonico, Andrea; Oh, Joo Yeon; Sobek, Jens; Heinemann, Matthias; Zenobi, Renato

doi:10.1002/anie.200705923

Cited by 136 publications

(126 citation statements)

References 36 publications

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“…Recently, the limits of detection for metabolites using mass spectrometry have been lowered from typically femtomoles to the low attomole range which is the range that is required for single cell metabolomics [30 ]. However, even if such small quantities can be detected, quantification is still problematic, and so is handling of the minute sample quantities (originating from an individual cell).…”

Section: Challenges For Single Cell Metabolomicsmentioning

confidence: 99%

“…[34]). Also, it has already been demonstrated for metabolites including UDP, ADP, GDP, UTP, ATP, GTP, acetyl-CoA, and butyryl/isobutyryl-CoA that the sensitivity for MALDI-based MS detection of metabolites will be sufficient to reach the single yeast cell level [30 ] and a suitable technology for sample deposition (''writing'' onto a MALDI plate) was also proposed [12,35,36] allowing for convenient off-line mass-spectrometric analysis. Coupling of the Figure 1 Schematics of the four MS-based approaches for single cell metabolomics.…”

Section: Ms-based Approaches For Single Cell Metabolomicsmentioning

confidence: 99%

“…Compared to the metabolite amounts that are typically used for classical populationlevel metabolomics (nanomoles), single cell metabolomics for E. coli has to handle amounts that are approximately 10 9 times lower. Unlike in single cell genomics and proteomics, amplification of analyte and/or highly sensitive fluorescence measurements on labelled compounds cannot be used for single cell metabolomics.Recently, the limits of detection for metabolites using mass spectrometry have been lowered from typically femtomoles to the low attomole range which is the range that is required for single cell metabolomics [30 ]. However, even if such small quantities can be detected, quantification is still problematic, and so is handling of the minute sample quantities (originating from an individual cell).…”

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Single cell metabolomics

Heinemann

Zenobi

2011

Current Opinion in Biotechnology

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117

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Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single cell metabolomics to investigate metabolic phenomena that are buried under the population average. Here, we review the current approaches and illustrate their advantages and disadvantages. Because of significant advances in the field, different technologies are now at the verge of generating data that are useful for exploring and investigating metabolic heterogeneity. IntroductionQuantitative metabolomics, the technology for large-scale quantification of intracellular metabolite concentrations, is a powerful tool in systems biology research that has recently led to a series of interesting findings (e.g. [1][2][3][4]). Because of the metabolome's chemical diversity, mass spectrometry (MS) is the analytical method of choice [5]. In addition to analytical challenges, quantitative metabolomics as required for addressing (systems) biology questions poses significant challenges in sample processing. One important challenge is the need to preserve the original metabolome during sample processing, which is often difficult because of the presence of enzymes in the sample and the fast metabolic turnover rates.For sensitivity reasons, current metabolomics methods require samples that contain a large number of cells. However, cell populations are not necessarily homogeneous. Besides genetic differences, several other sources for population heterogeneity exist, of which several are also known to cause metabolic differences. Today, methods capable of resolving differences in metabolite levels on the single cell level are provided, within limits, by molecular sensors such as FRET sensors [6,7] or aptamer-based technology [8 ,9]. Both types of molecular sensors, however, are difficult to develop, are limited to specific analytes, and quantitative analyses (e.g. in terms of mol/L) are hardly possible with them. Laserinduced fluorescence, as introduced by Dovichi for single cell proteomics, is limited to fluorescent compounds or labelled species [10,11]. In addition, all these existing methods share the limitation that they can never be extended to the ''-omics'' level, that is to measuring a large number of metabolites at the same time. They will thus not be applicable to discovery type research and research that requires a large number of metabolites to be measured in the same cell.Because of the success of mass spectrometry in population-level metabolome analyses, the analytical community has recently made great strides towards single cell level metabolite analyses (for a review, see [12]). So far, however, hardly any new biological insight has been generated from these endeavours. In this Current Opinion paper, we will thus not only review the current status of MS-based single cell metabolomics but also discuss which of the differ...

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Section: Challenges For Single Cell Metabolomicsmentioning

confidence: 99%

Section: Ms-based Approaches For Single Cell Metabolomicsmentioning

confidence: 99%

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confidence: 99%

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Single cell metabolomics

Heinemann

Zenobi

2011

Current Opinion in Biotechnology

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117

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“…Although recent advances in mass spectroscopy have allowed to reach single cell detection levels for some proteins or metabolites 10,11 , this technique is not yet generally used to analyze signal transduction pathways but might add, in the future, a different approach to analyze the content of single cells. For this chapter, we will focus our discussion on the experimental methods that currently offer single cell resolution which are optical methods such as microscopy and flow cytometry.…”

Section: Fluorescent-based Quantitative Measurements Of Signal Transdmentioning

confidence: 99%

Fluorescent-Based Quantitative Measurements of Signal Transduction in Single Cells

Pelet¹,

Peter²

2011

Design and Analysis of Biomolecular Circuits

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Budding yeast (Saccharomyces cerevisiae) has been widely used as a model system to study fundamental biological processes. Genetic and biochemical approaches have allowed in the last decades to uncover the key components involved in many signaling pathways. Generally, most techniques measure the average behavior of a population of cells, and thus missed important cell-to-cell variations. With the recent progress with fluorescent proteins, new avenues have been opened to quantitatively study the dynamics of signaling in single living cells. In this chapter, we describe several techniques based on fluorescence measurements to quantify the activation of biological pathways. Flow cytometry allows for rapid quantification of the total fluorescence of a large number of single cells. In contrast, microscopy offers a lower throughput but allows to follow with a high temporal resolution the localization of proteins at sub-cellular resolution. Finally, advanced functional imaging techniques such as FRET and FCS offer the possibility to directly visualize the formation of protein complexes or to quantify the activity of proteins in vivo. Together these techniques present powerful new approaches to study cellular signaling and will greatly increase our understanding of the regulation of signaling networks in budding yeast and beyond. FLUORESCENT-BASED QUANTITATIVE MEASUREMENTS OF SIGNAL TRANSDUCTION IN SINGLE CELLS Serge Pelet and Matthias Peter Institute of Biochemistry, Department of Biology, ETH Zürich Abstract:Budding yeast (Saccharomyces cerevisiae) has been widely used as a model system to study fundamental biological processes. Genetic and biochemical approaches have allowed in the last decades to uncover the key components involved in many signaling pathways. Generally, most techniques measure the average behavior of a population of cells, and thus missed important cell-tocell variations. With the recent progress with fluorescent proteins, new avenues have been opened to quantitatively study the dynamics of signaling in single living cells. In this chapter, we describe several techniques based on fluorescence measurements to quantify the activation of biological pathways. Flow cytometry allows for rapid quantification of the total fluorescence of a large number of single cells. In contrast, microscopy offers a lower throughput but allows to follow with a high temporal resolution the localization of proteins at sub-cellular resolution. Finally, advanced functional imaging techniques such as FRET and FCS offer the possibility to directly visualize the formation of protein complexes or to quantify the activity of proteins in vivo.Together these techniques present powerful new approaches to study cellular signaling and will greatly increase our understanding of the regulation of signaling networks in budding yeast and beyond.

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“…In spite of these obstacles, Kennedy and coworkers, for example, developed an extremely useful MALDI method for metabolome analysis by choosing an appropriate chemical matrix (Edwards and Kennedy 2005;Sun et al 2007). This method was further optimized to reach a sensitivity capable even of detecting the metabolites of a single yeast cell (Amantonico et al 2008). Several other MALDI methods tailored to specific analytes classes have been developed trough careful optimization of the sample preparation and proper choice of the matrix (Cohen and Gusev 2002).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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 demonstrated.

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Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

Cited by 136 publications

References 36 publications

Single cell metabolomics

Single cell metabolomics

Fluorescent-Based Quantitative Measurements of Signal Transduction in Single Cells

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

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