Single Cell Analysis with Probe ESI-Mass Spectrometry: Detection of Metabolites at Cellular and Subcellular Levels

Abstract

Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 μm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell "omics".

Fast Quantitation of Target Analytes in Small Volumes of Complex Samples by Matrix‐Compatible Solid‐Phase Microextraction Devices

“…[1][2][3][4] Facilitated by the improved sensitivity and specificity provided by modern mass spectrometry (MS) instrumentation, the replacement of traditional complex laboratory procedures with integrated miniaturized methods has become ag rowing trend in POC diagnosis. [5] In recent years,advances in direct sample to mass spectrometry techniques,s uch as paper-spray ionization, probe electrospray ionization, and touch spray [2,[5][6][7] have allowed for the application of these methods towards the quantitative analysis of small volumes of biofluids.However, the sensitivity and precision typically achieved in the laboratory through adequate sample preparation prior to the MS quantification is traded off.T herefore,techniques capable of isolating and enriching target analytes from complex matrices with minimal processing time and adequate sample clean-up are highly desirable for applications that require direct introduction to MS. [1-3, 8, 9] As ac oncept, solid-phase microextraction (SPME) embraces solventless microextraction technologies with different geometrical configurations that efficiently integrate sampling and sample clean-up,while also allowing for enrichment of the molar fraction of ag iven analyte in as ingle step. [10] Given the multiple advantages of this technique, including its feasibility to be coupled to different analytical instruments,S PME has been widely used for analysis of complex matrices such as biofluids,t issues,a nd food samples.…”

“…single cells [13] ), but can also diminish potential damage caused during in vivo sampling. [9] Although the use of small metal probes for the analysis of single cells and tissue has already been reported, [6,13] owing to the low sorption capacity and non-specific affinity of the metal surface (poor inter-device reproducibility), their application is limited to the determination of compounds present at high concentrations (e.g. phospholipids).…”

“…Unlike other reported approaches that are based on metal needles without coating, [6,7,19] SPME acts as achemical biopsy tool by enriching small molecules carrying chemical information about the investigated system without removing tissue,t hus providing am uch cleaner extraction. Currently,o ur group is working on the development of versatile biocompatible coatings for miniature devices that allow for the extraction of abroader range of compounds,with potential for avariety of applications in fields,s uch as plant biology and oncology, where small objects need to be scrutinized.…”

Desorption in Mass Spectrometry

“…87) Metabolites loaded on the tip surface were extracted by the auxiliary solvent generated by a piezo-electric microdroplet generator and electrosprayed a er applying a high voltage. Gong et al detected metabolites at cellular and subcellular levels by PESI-MS. 88) Tungsten probes with a tip diameter of ∼1 µm were inserted into live A. cepa (onion) cells. By spraying the solvent vapor to the needle tip, metabolites were electrosprayed from the tip of the probe.…”

Development of Novel Solid-Phase Microextraction Fibers

“…Gong et al [170] used tungsten wires with the tip diameters of about 1 μm to sample metabolites in single Allium cepa cells. Subcellular analysis was realized in nucleus and cytoplasm.…”