Metabolites and peptides play important roles in almost every aspect of cell function. Their intracellular levels and spatial localizations reflect the state of each cell and its relationship to its surrounding environment. Moreover, their levels and dynamics are indicative of normal or pathological cellular conditions. Bioanalytical technologies for microanalysis are able to qualitatively and quantitatively characterize subsets of peptides and metabolites from individual microorganism, plant and animal cells. Highlighted here are the established and evolving strategies for characterization of the metabolome and peptidome of single cells. Focused studies of the chemical composition of individual cells and their networks promise to provide a greater understanding of cellular fate, function, and homeostatic balance. Single cell bioanalytical microanalysis has also become increasingly valuable for examining cellular heterogeneity, particularly in the fields of neuroscience, stem cell biology, and developmental biology.

The feasibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging of features smaller than the laser beam size has been demonstrated. The method involves the complete ablation of the MALDI matrix coating the sample at each sample position and moving the sample target a distance less than the diameter of the laser beam before repeating the process. In the limit of complete sample ablation, acquiring signal from adjacent positions spaced by distances smaller than the sample probe enhances image resolution as the measured analyte signal only arises from the overlap of the laser beam size and the non-ablated sample surface. [3] has revolutionized the investigation of biological molecules by providing soft ionization methods linking biochemistry with the powerful analysis tools of mass spectrometry (MS). In the analysis of complex samples, such as biological tissue, MALDI is of particular interest because of its ability to desorb and ionize molecules of high molecular weight, such as proteins and peptides, providing excellent sensitivity while retaining considerable tolerance towards salts and other small molecules found at high concentration in tissue. It has been roughly 10 years since the first published applications in which MALDI-MS was used to create a chemical images of substrates [4,5]. The intervening decade has seen a considerable growth in techniques and instrumentation for MALDI-MS imaging, developed largely by Caprioli and coworkers [6 -12] with contributions to sampling techniques [13][14][15][16] and instrumentation [17,18] provided by others.It is generally accepted that the maximal spatial imaging or profiling resolution of microprobe imaging techniques is determined by a combination of the size of the microprobe and the precision of the sample or microprobe positioning device. MALDI mass spectrometers typically use lasers having relatively large beam sizes (about 100 m diameter) in the analysis of standard, dried-droplet preparations. Some effort has been put into decreasing the size of the laser beam sizes for MALDI-MS imaging and profiling of biological samples, particularly for samples containing a high proportion of peptidergic neurons or other secretory cells. Investigated biological samples of this nature include rat pituitary and rat pancreas [6], mouse brain and human brain tumor xenografts [8,14], rat brain and rat brain tumors [9,19], mouse epididymis [20], molluscan atrial gland [15], and molluscan peptidergic neurons [16].Ideally, the spatial resolution of MALDI-MS imaging of analyte-rich tissues would approach the size of a single mammalian cell, 5 to 20 m in diameter. Several strategies have been used or suggested to decrease the laser beam diameter in imaging applications, including the placement of a pinhole aperture between the outlet of the laser and the focusing optics of the mass spectrometer [6,13], decreasing the size of the fiber optic used to direct the laser into the MALDI source [9], and placing multiple lenses between the laser and the M...

A method that enables metabolomic profiling of single cells and subcellular structures is described using capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry. A nebulizer-free coaxial sheath-flow interface completes the circuit and provides a stable electrospray, yielding a signal with a relative standard deviation of under 5% for the total ion electropherogram. Detection limits are in the low nanomolar range (i.e., < 50 nM (< 300 amol)) for a number of cell-to-cell signaling molecules, including acetylcholine (ACh), histamine, dopamine, and serotonin. The instrument also yields high efficiency separations, e.g., ~600,000 for eluting ACh bands. The utility of this setup for single cell metabolomic profiling is demonstrated with identified neurons from Aplysia californica-the R2 neuron and metacerebral cell (MCC). Single cell electropherograms are reproducible, with a large number of metabolites detected; more than 100 compounds yield signals of over 10 4 counts from the injection of only 0.1% of the total content from a single MCC. Expected neurotransmitters are detected within the cells (ACh in R2 and serotonin in MCC), as are compounds that have molecular masses consistent with all of the naturally-occurring amino acids (except cysteine). Tandem MS using a quadrupole time-of-flight tandem mass spectrometer distinguishes ACh from isobaric compounds in the R2 neuron and demonstrates the ability of this method to characterize and identify metabolites present within single cells.

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Vitamin E (alpha-tocopherol) has been implicated in several cellular processes including signaling, transport, lipid membrane curvature, and several neurodegenerative disorders. Vitamin E imaging has been hindered by the inaccessibility of the molecule to traditional immunohistochemical methods. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS), the distribution of major constituents in the cellular membrane of isolated neurons was investigated. There is a significant increase in the vitamin E signal at the soma-neurite junction compared to the cell as a whole (165 +/- 11% of that found across the cell, p = 0.004, n = 12). The observed membrane distribution suggests an important new role for vitamin E in neuronal function.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is used for the multiplex detection and characterization of diverse analytes over a wide mass range directly from tissues. However, analyte coverage with MALDI MSI is typically limited to the more abundant compounds, which have m/z values that are distinct from MALDI matrix-related ions. On-tissue analyte derivatization addresses these issues by selectively tagging functional groups specific to a class of analytes, while simultaneously changing their molecular masses and improving their desorption and ionization efficiency. We evaluated electrospray deposition of liquid-phase derivatization agents as a means of on-tissue analyte derivatization using 2-picolylamine; we were able to detect a range of endogenous fatty acids with MALDI MSI. When compared with airbrush application, electrospray led to a 3-fold improvement in detection limits and decreased analyte delocalization. Six fatty acids were detected and visualized from rat cerebrum tissue using a MALDI MSI instrument operating in positive mode. MALDI MSI of the hippocampal area allowed targeted fatty acid analysis of the dentate gyrus granule cell layer and the CA1 pyramidal layer with a 20-μm pixel width, without degrading the localization of other lipids during liquid-phase analyte derivatization.

MALDI MS imaging and single-cell profiling are important new capabilities for mass spectrometry. The distribution of neuropeptides within a cell plays an important role in the functioning of the cells in a neuronal network. Protocols for subcellular MALDI MS are described that allow comparative peptide profiling of cell bodies and the neuronal processes (neurites) using single isolated neurons from the neuronal model Aplysia californica. The seawater surrounding the neurons is problematic for mass spectrometry and so must be removed in a manner that does not cause morphological changes or a redistribution of the neuropeptides. Several protocols have been investigated for subcellular spatial profiling, including the use of air-drying, replacement of the seawater with deionized water, and substitution of the cell matrix with fluorinert, mineral oil and glycerol, as well as paraformaldehyde fixation. Glycerol stabilization offers the best combination of preservation of cell morphology and prevention of neuropeptide redistribution. The profiles of the peptides in specific neuronal processes and the cell bodies demonstrate a variety of differences that appear to be cell-specific. These methods are suitable for smaller cells and subcellular MS imaging.

Single cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15), chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.