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.

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In the search for a more effective adjuvant therapy to treat multiple myeloma (MM), we investigated the effects of the traditional Chinese herbal medicines Huang-Lian-Jie-Du- Tang IntroductionMultiple myeloma (MM) is an incurable plasma-cell malignancy and the second most common hematologic malignancy, with 14 000 new patients diagnosed in the United States annually. 1,2 Although combination chemotherapy offers initial response rates of 40% to 70% in MM patients, 3 refractoriness to these regimens eventually develops. High-dose chemotherapy with stem cell support has achieved higher response rates than conventional therapy, but few patients remain in long-term remission. 4 Thus, the development of a more effective therapy to treat early and advanced MM has become a priority.Many components from herbs have been identified as effective in the treatment of human disease. Curcumin, a major component of turmeric, is able to correct defects associated with the homozygous expression of delta F508 cystic fibrosis 5 and to suppress the growth of myeloma cells. 6 Arsenic trioxide, a compound of arsenic, is very effective in the treatment of patients with acute promyelocytic leukemia who have developed resistance to all-trans retinoic acid (ATRA). 7 Artemisinins, extracted from sweet wormwood, are the most potent antimalarials available, rapidly killing Plasmodium falciparum at all asexual stages by inhibiting the sarcoplasmic or endoplasmic reticulum calcium ATPase (SERCA) ortholog (PfATP6) in Xenopus oocytes with a similar potency to thapsigargin. 8 Consequently, they are widely used to treat multidrug-resistant malaria, a disease that claims 1 million lives annually. 9 Inflammation and MM may be induced partly in the same way, as interleukin 6 (IL-6) is a potential mediator in these conditions. 10,11 Many Kampo medicines have been used historically in anti-inflammatory therapy. By screening the effects of antiinflammatory Kampo formulas on MM cells, we hoped to find one to treat MM. Huang-Lian-Jie-Du-Tang (HLJDT) contains Coptis rhizoma, Phellodendron bark, Scutellaria radix (root), and Gardenia fruit in 2.0, 1.5, 3.0, and 2.0 parts, respectively. It is recognized in Japan and China as an effective anti-inflammatory agent and has been widely used in the treatment of various inflammatory diseases such as gastritis, dermatitis, aphthous stomatitis, and hypertension. HLJDT exhibited anti-inflammatory activity in experimental colitis induced by dextran sulfate sodium, 12 and in animal experiments it inhibited the proliferation of lymphocytes under inflammatory conditions by suppressing the secretion of proinflammatory cytokines including interferon ␣ (IFN-␣) and IFN-␥. 13 The secretion of these cytokines was also reported to be suppressed by HLJDT in is the recipient of a Postdoctoral Fellowship Award for Foreign Researchers (P04500) from the Japan Society for the Promotion of Science (JSPS).Reprints: Michio M. Kawano, Department of Bio-Signal Analysis, AMES, Graduate School of Medicine, Yamaguchi University, 1-1-1 Minami-...

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...