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.

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.

Rh-catalyzed direct carboxylation of unactivated aryl C-H bond under atmospheric pressure of carbon dioxide was realized via chelation-assisted C-H activation for the first time. Variously substituted and functionalized 2-arylpyridines and 1-arylpyrazoles underwent the carboxylation in the presence of the rhodium catalyst and a stoichiometric methylating reagent, AlMe(2)(OMe), to give carboxylated products in good yields. The catalysis is proposed to consist of methylrhodium(I) species as the key intermediate, which undergoes C-H activation to afford rhodium(III), followed by reductive elimination of methane to give nucleophilic arylrhodium(I). This approach demonstrates promising application of C-H bond activation strategy in the field of carbon dioxide fixation.

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

When we are able to analyze molecules of visualized reacting cells directly in real time, studies of molecular mechanisms of living systems will become more direct and fast. However, the response of cells to stimuli is not the same, but slightly different from cell to cell. We should thus seek a very sensitive and exhaustive molecular detection method for a single cell with simultaneous video-microscopic observation. Bioimaging is very useful to visualize the distribution and dynamism of probed molecules or ions in a cell.2,3 However, it is incapable of finding new molecules. On the other hand, mass spectrometry (MS) can detect the existence of both known and unknown molecules, and has been widely applied to current biological molecular analyses, such as imaging of various molecular distributions in tissues, 4,5 metabolomics, 6,7 and proteomics. 8 Single-cell MALDI-TOF/MS analysis 9,10 is an approximate study, but the number of detected MS peaks were fewer than expected in a cell. 11 We have developed the method to detect hundreds to thousands of small molecular MS peaks from a living single cell in order to extract and identify the key molecules contained specifically in a cell. 12 An adherent cell line, mouse embryonic fibroblasts Swiss 3T3, was cultured in Dulbecco's modified Eagle minimal essential medium supplemented with 10% fetal calf serum (FCS), 100 mg/mL penicillin and 100 mg/mL streptomycin G in 5% CO2 at 37˚C. 13 Cultured cells were monitored by a CCD video camera mounted on an inverted microscope (OLYMPUS, IX-70). The cytoplasm contents of a target cell, viewed by a video microscope, was sucked into a gold-coated glass capillary nanoelectrospray tip (Humanix, nanospray tip, Japan) set on a micromanipulator (Narishige, MHW-103, Japan) using a connected syringe via tubing. For positive-mode detection, acetonitrile containing 0.5% formic acid was added as an ionization solvent to the sucked sample solution in the capillary nanospray tip.Mass spectrometric detection was performed by a Q-TOF mass spectrometer (Applied Biosystems, QSTAR-XL) equipped with a nano-ESI ion source. The spray voltage was set to around 1000 V. Calibration was performed at every hour and at the beginning of each measurement using dopamine; m/z 137.0597 + and angiotensin I; m/z 432.8998The intensities of MS peaks were normalized by the intensity of a solvent peak at m/z 381.26. MS/MS analysis was performed with a collision energy from 10 to 30 eV. The obtained spectra were analyzed by Markerview (Applied Biosystems) software for a t-test analysis. A nano-electrospray ionization (ESI) tip was directly inserted into a single cell of Swiss 3T3 as shown in Fig. 1(a) to suck its cytoplasmic contents, and was then set to a nano-ESI attachment of a mass spectrometer for molecular ionization. Hundreds of MS peaks of small molecules out of less than a 1-pl sample were successfully detected ( Fig. 1(b)). Comparatively, the MS spectra of cell incubating medium (Fig. 1(c)) and the ionization solvent ( Fig. 1(d)) were measured in the same way...

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Summary Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell‐specific TIAs localisation and accumulation with 10 μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single‐cell mass spectrometry (single‐cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Direct carboxylation of simple arenes under atmospheric pressure of CO2 is achieved through a rhodium-catalyzed C-H bond activation without the assistance of a directing group. Various arenes such as benzene, toluene, xylene, electron-rich or electron-deficient benzene derivatives, and heteroaromatics are directly carboxylated with high TONs.