Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.
The molecular content from the cytoplasm of a live, single mammalian cell and its organelle were trapped with a nano-electrospray ionization (ESI) tip acting as a micropipette under a video microscope, and hundreds of small molecular peaks were detected by direct nano-ESI mass spectrometry (MS). Granule- or cytoplasm-specific peaks in a mast cell (RBL 2H3) model were extracted by paired t-test to demonstrate their specific localization. Some of the typical and specific molecules were successfully identified by MS/MS analysis. This method was also applied to the cell classification of seven types of cell lines at the single-cellular level by principal component analysis (PCA), revealing seven clusters in the multivariate score plot.
The history from bio-imaging to live single-cell mass spectrometry (MS) is herein reviewed. The limitation of the current bio-imaging method is probing only known molecules, and a method for finding new molecules is needed for cells which, however, show individual behaviors even in the same incubation dish. Single-cell MALDI-TOF/MS has been developed, but it can detect only molecules that can be easily ionized, and not be exhaustive. Recently, the contents of a single cell have been sucked out by a nano-electro spray tip, and directly introduced into MS by nano-spray ionization. Thousands of molecular peaks have been successfully and exhaustively detected, and an extraction method for key molecules was also developed. This new method is now being widely applied to explore site-or state-specific molecules in various aspects of cell dynamisms.
Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue. A lkaloids constitute one of the largest groups of specialized metabolites, many of which have biological functions that are indispensable, not only for plants themselves but also for human health. Approximately 20% of plant species are known to contain alkaloids (1). The significant value of alkaloids as medicines or luxury items in human life has attracted widespread interest from researchers in a range of scientific fields. These researchers have extensively studied how plant-specialized metabolites are produced at cellular and tissue levels (2). The reports indicate that biosynthetic pathways of plant specialized metabolites often involve multiple cell types that are biochemically and morphologically distinct (3,4
Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.
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