Mass spectrometry-based metabolomics of single yeast cells

Abstract

Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study, we demonstrate that, with the microarrays for mass spectrometry platform, we are able to observe this variability. We monitor environmentally (2-deoxy-D-glucose) and genetically (ΔPFK2) perturbed Saccharomyces cerevisiae cells at the single-cell, few-cell, and population levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge, give biological insight that is not accessible from population-level metabolomic data.single-cell measurements | MALDI mass spectrometry | baker's yeast E ven genetically identical cells present in the same microenvironment can express different phenotypes, for a number of reasons: cell-to-cell heterogeneity can stem from differences in the cell age and differences in the cell cycle stage, and stochastic effects together with feedback mechanisms can lead to distinctively different phenotypes, too (1-6). As population-level measurement techniques inherently average out such cell-to-cell differences, biochemical mechanisms underlying a studied system cannot be deduced from such measurements. Thus, to detect and exploit this heterogeneity, new analytical platforms with a sensitivity at the single-cell level and the ability to perform quantitative analyses must be developed and validated.Motivated by advances of mass spectrometry (MS) in metabolomics, the analytical chemistry community has stepped up its efforts toward realizing MS-based single-cell metabolomics (1, 2). A number of analytical approaches were developed with detection limits low enough for single-cell metabolite analyses [e.g., nanostructured surfaces (7, 8), postionization techniques (9, 10), modified laser optics (11), the use of microsampling tools (12, 13), microarrays for MS measurements (14, 15), etc.]. Until now, however, most MS studies targeting single-cell metabolite analysis have only shown the analytical capabilities, but have not demonstrated that true biological information can be retrieved from studying the metabolism of single cells.Here, using the unicellular eukaryotic model organism Saccharomyces cerevisiae, we present an analytical validation of a single-cell metabolite analysis using the microarrays for mass spectrometry (MAMS) platform. This validation concerns both the analytical methodology and the biological information, by monitoring expected cellular responses upon an environmental and a genetic perturbation. Furthermore, we present examples of biological insight that are only accessible with a platform such as MAMS. Specifically, we unravel metabolite-metabolite correlations, and visualize coexisting subpopulations in an isogenic cell culture. This technology can now be used to reveal metabolic differences in cells of isogenic cell populations, such as differences caused by cell cycles stages, cell ages, or stochastically induced phenotypic differences. Results and ...

Screening of Chlamydomonas reinhardtii Populations with Single-Cell Resolution by Using a High-Throughput Microscale Sample Preparation for Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

“…Furthermore, the selectivity of tags is often determined by the strength of noncovalent interactions specific to an analyte, which can render labeling strategies highly complex. Low-molecular-weight compounds often lack enough distinctive binding motifs to allow for specific binding in complex cellular environments.On the other hand, some analytes (e.g., molecules with structural functions, such as lipids) are present in much higher copy numbers inside cells than are DNA or proteins, such that they are within the reach of state-of-the-art mass spectrometric detection, as was shown in recent years (11,12). Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry is the method of choice for the analysis of tissues (13), and new laser desorption sources render it possible to image the distribution of small molecules with single-cell resolution (14,15).…”

“…On the other hand, some analytes (e.g., molecules with structural functions, such as lipids) are present in much higher copy numbers inside cells than are DNA or proteins, such that they are within the reach of state-of-the-art mass spectrometric detection, as was shown in recent years (11,12). Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry is the method of choice for the analysis of tissues (13), and new laser desorption sources render it possible to image the distribution of small molecules with single-cell resolution (14,15).…”

Metabolic profiling in diabetes

“…Here again, a combination of deep metabolic phenotyping with large sample numbers, including biological replicates, multiple conditions, and time points, are key to success. In contrast to studies on human samples, animal studies allow for access to a combination of tissues from several organs, while work on cell culture allows for the variation of a single parameter at a time and potentially even investigation of the metabolome of a single cell (Ibanez et al 2013), possibly limited to specific subcellular organelles. Moreover, although only briefly mentioned in this review, the value of parallel access to complementary large-scale data, such as genome-wide genetic variation, DNA methylation, gene expression, noncoding RNA levels, and proteomics cannot be overestimated.…”

Metabolomic Analysis of Schizosaccharomyces pombe: Sample Preparation, Detection, and Data Interpretation

“…Another interesting topic is the analysis of metabolic fluxes, also called fluxomics (Mo et al 2009;Celton et al 2012). In budding yeast, the first single-cell metabolomic studies have recently appeared (Ibanez et al 2013;Zenobi 2013), and methods for highthroughput screening of low-volume cultures from 96-well plates are also being developed (Ewald et al 2009 …”