Alfredo J. Ibáñez

Mass spectrometry-based metabolomics of single yeast cells

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.

Cytosolic pH Regulates Cell Growth through Distinct GTPases, Arf1 and Gtr1, to Promote Ras/PKA and TORC1 Activity

Regulation of cell growth by nutrients is governed by highly conserved signaling pathways, yet mechanisms of nutrient sensing are still poorly understood. In yeast, glucose activates both the Ras/PKA pathway and TORC1, which coordinately regulate growth through enhancing translation and ribosome biogenesis and suppressing autophagy. Here, we show that cytosolic pH acts as a cellular signal to activate Ras and TORC1 in response to glucose availability. We demonstrate that cytosolic pH is sensitive to the quality and quantity of the available carbon source (C-source). Interestingly, Ras/PKA and TORC1 are both activated through the vacuolar ATPase (V-ATPase), which was previously identified as a sensor for cytosolic pH in vivo. V-ATPase interacts with two distinct GTPases, Arf1 and Gtr1, which are required for Ras and TORC1 activation, respectively. Together, these data provide a molecular mechanism for how cytosolic pH links C-source availability to the activity of signaling networks promoting cell growth.

Analysis of single algal cells by combining mass spectrometry with Raman and fluorescence mapping

In order to investigate metabolic properties of single cells of freshwater algae (Haematococcus pluvialis), we implement matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with microspectroscopic mapping. Straightforward coupling of these two detection platforms was possible thanks to the self-aliquoting properties of micro-arrays for mass spectrometry (MAMS). Following Raman and fluorescence imaging, the isolated cells were covered with a MALDI matrix for targeted metabolic analysis by MALDI-MS. The three consecutive measurements carried out on the same cells yielded complementary information. Using this method, we were able to study the encystment of H. pluvialis - by monitoring the adenosine triphosphate (ATP) to adenosine diphosphate (ADP) ratio during the build-up of astaxanthin in the cells as well as the release of β-carotene, the precursor of astaxanthin, into the cytosol.

Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis.

BackgroundSuccessful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.ResultsInfrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response.A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings.ConclusionThe described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids, oxylipins, and carbohydrates. Moreover, potential novel biomarkers can be detected and used to predict the quality of plant infections.

Carbon-13 labelling strategy for studying the ATP metabolism in individual yeast cells by micro-arrays for mass spectrometry

Isotopic labelling of cellular metabolites, used in conjunction with high-density micro-arrays for mass spectrometry enables observation of ATP metabolism in single yeast cells.

Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in 13C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

DNA detection using a triple readout optical/AFM/MALDI planar microwell plastic chip

A ready-to-spot disposable DNA chip for specific and sensitive detection of DNA was developed. Plastic copolymeric substrate chemistry was optimized to selectively couple the target DNA with the active chip surface. At the same time, the developed substrate limits the unspecific adsorption of probe DNA molecules or additional polar contaminants in the test samples to the chip surface. The combination of glycidyl and n-butyl methacrylates was found to best fit the requirements of the assay. The fabricated DNA microarrays have mechanical properties similar to those of the glass or silicon substrates and, at the same time, provide chemically reactive surfaces that do not require lengthy chemical modification. An additional advantage of the plastic microchip is its compatibility with different analytical readout techniques, such as mass spectrometry (MALDI-TOF/MS), optical detection (fluorescence and enzyme-induced metal deposition), and imaging techniques (atomic force microscopy). These multiple readout techniques have given us the ability to compare the sensitivity, selectivity, and robustness of current state-of-the-art bioanalytical methods on the same platform exemplified by successful DNA-based detection of human cytomegalovirus. The obtained sensitivity for enzymatically enhanced silver deposition (10(-15) M) surpasses that of conventional fluorescence readouts. In addition, the assays dynamic range (10(-6)-10(-15) M), reproducibility, and reliability of the DNA probe detection speaks for the silver deposition method. At compromised sensitivity (10(-9) M), the length of the DNA probes could be checked and, alternatively, DNA single point polymorphisms could be analyzed.

Analysis of metabolites in single cells-what is the best micro-platform?

This review covers new innovations and developments in the field of single‐cell level analysis of metabolites, involving the role of microfluidic and microarray platforms to manipulate and handle the cells prior their detection. Microfluidic and microarray platforms have shown great promise. The latest developments demonstrate their potential to identify a particular cell or even an ensemble of cells (sharing a common property or phenotype) that co‐exist in a much larger cell population. The reason for this is the capability of these platforms to perform several complex analytical processes, such as: cleanup, sorting, derivatization, separation, and detection, with great robustness, speed, and reduced sample/reagent consumption. Here, we present several examples that illustrate the rapid strides that have been made for the routine analysis of metabolites by coupling different microfluidics and microarrays devices to a wide range of analytical detectors (e.g. fluorescent microscopy, electrochemical, and mass spectrometry). Herein, we also present selected examples detailing the use of microfluidics and microarrays in the visualization of the natural occurring cell‐to‐cell heterogeneity in isogenic populations, in particular during the response to external cues. The possibility to accurate monitor the cell‐to‐cell heterogeneity based on different levels of key metabolites is of clinical relevance, since cell‐to‐cell heterogeneity can influence, for example, the outcome of a drug treatment.

Capturing in Vivo Plant Metabolism by Real-Time Analysis of Low to High Molecular Weight Volatiles.

We have deployed an efficient secondary electrospray ionization source coupled to an Orbitrap mass analyzer (SESI-MS) to investigate the emissions of a Begonia semperflorens. We document how hundreds of species can be tracked with an unparalleled time resolution of 2 min during day-night cycles. To further illustrate the capabilities of this system for volatile organic compounds (VOCs) analysis, we subjected the plant to mechanical damage and monitored its response. As a result, ∼1200 VOCs were monitored displaying different kinetics. To validate the soundness of our in vivo measurements, we fully characterized some key compounds via tandem mass spectrometry (MS/MS) and confirmed their expected behavior based on prior gas chromatography/mass spectrometry (GC/MS) studies. For example, β-caryophyllene, which is directly related to photosynthesis, was found to show a periodic day-night pattern with highest concentrations during the day. We conclude that the capability of SESI-MS to capture highly dynamic VOC emissions and wide analyte coverage makes it an attractive tool to complement GC/MS in plant studies.

Matrix-assisted laser desorption/ionization matrices for negative mode metabolomics.

Matrix-assisted laser desorption/ionization (MALDI) has been shown to be highly sensitive for analyzing low-mass compounds such as metabolites if the right matrix is used. 9-aminoacridine (9AA) is the most commonly employed matrix for negative mode MALDI-MS in metabolomics. However, matrix interferences and the strongly varying sensitivity for different metabolites make a search for alternative matrices desirable, in order to identify compounds with a different chemical background and/or favoring a different range of analytes. We tested the performance of a series of potential negative mode MALDI matrices with a mix of 29 metabolites containing amino acids, nucleotide phosphates and Krebs cycle intermediates. While ethacridine lactate was found to provide limits of detection (LODs) in the low femtomole range for nucleotide phosphates, amino acids and Krebs cycle intermediates in the low picomole range, 4-amino-2-methylquinoline showed LODs in the picomole range for most metabolites, but is capable of ionizing a broader range of analytes than both 9AA and ethacridine.