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.

Multidimensional analysis of single algal cells by integrating microspectroscopy with mass spectrometry.

We demonstrate a facile label-free approach for performing multidimensional chemical analysis on individual single-cell organisms by combining optical, fluorescence, and Raman microspectroscopy with matrix-free laser desorption/ionization mass spectrometry (MS). Single unicellular algae are seeded on a bare stainless steel plate and analyzed microspectroscopically. This provides information on the content and distribution of photoactive species, such as ß-carotene, as well as chlorophyll and other components of the photosynthetic apparatus. Exactly the same cells are then analyzed by mass spectrometry in the negative ion mode. Phospholipid species are readily ionized by laser desorption/ionization of intact cells, without the need for an auxiliary matrix. This not only facilitates sample preparation but also preserves high spatial resolution and high sensitivity. Using this method, we were able to study the content and arrangement of proplastids and photosystem components, as well as the amounts of various phospholipid species in individual algal cells. The methodology can be used in the fundamental biological studies on these unicellular organisms, which require information on the internal structure as well as the chemical composition of individual cells.

Lab-on-a-plate: extending the functionality of MALDI-MS and LDI-MS targets.

We review the literature that describes how (matrix-assisted) laser desorption/ionization (MA)LDI target plates can be used not only as sample supports, but beyond that: as functional parts of analytical protocols that incorporate detection by MALDI-MS or matrix-free LDI-MS. Numerous steps of analytical procedures can be performed directly on the (MA)LDI target plates prior to the ionization of analytes in the ion source of a mass spectrometer. These include homogenization, preconcentration, amplification, purification, extraction, digestion, derivatization, synthesis, separation, detection with complementary techniques, data storage, or other steps. Therefore, we consider it helpful to define the "lab-on-a-plate" as a format for carrying out extensive sample treatment as well as bioassays directly on (MA)LDI target plates. This review introduces the lab-on-plate approach and illustrates it with the aid of relevant examples from the scientific and patent literature.

High-density micro-arrays for mass spectrometry.

Functional high-density micro-arrays for mass spectrometry enable rapid picolitre-volume aliquoting and ultrasensitive analysis of microscale samples, for example, single cells.

Miniature flowing atmospheric-pressure afterglow ion source for facile interfacing of CE with MS.

Here, we present a miniaturized version of the flowing atmospheric-pressure afterglow (miniFAPA) ion source and use it for sheathless coupling of CE with MS. The simple design of the CE-miniFAPA-MS interface makes it easy to separate the electric potentials used for CE and for ionization. A pneumatically assisted nebulization of the CE effluent transfers the analytes from the liquid phase into the gas phase before they are ionized by interacting with reactive species produced by the FAPA. An important advantage of this interface is its high stability during operation: optimization of five different parameters indicated that the interface is not sensitive to minor deviations from the optimum values. Other advantages include ease of construction and maintenance, as well as relatively low cost. Samples with complex matrices, such as yeast extract, soil extract and urine, spiked with the test compounds, were successfully analyzed using the CE-miniFAPA-MS setup.

Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

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

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 metabolomics.

Facile analysis of metabolites by capillary electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry using target plates with polysilazane nanocoating and grooves.

We present an inexpensive method and apparatus for the deposition of analytes separated by capillary electrophoresis (CE) onto a custom-designed matrix-assisted laser desorption/ionization (MALDI) target plate. This dedicated CE-MALDI plate is coated with an omniphobic polysilazane nanocoating and has an array of parallel grooves acting as recipients of the separation effluent. The 3-D pattern in the top layer of the coated plate greatly improves loading of the matrix solution prior to separation and facilitates deposition of the separated species. We demonstrate application of this straightforward protocol to the analysis of metabolites from the central metabolic pathway in a complex biological sample spiked with small molecule standards.