Live Single-Cell Molecular Analysis by Video-Mass Spectrometry

Abstract

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...

Fluorescence Probing Live Single-cell Mass Spectrometry for Direct Analysis of Organelle Metabolism

“…We have thus developed methods to capture single vacuoles and single granules. [1][2][3]11 When we combined live fluorescence imaging techniques with live single-cell and organelle mass spectrometry, we found we can trap even mitochondria. Mitochondria are a very important target in biology because of the important roles they play.…”

“…[1][2][3] A general protocol for plant cells has been published recently. 4 However, in addition to morphological information, fluorescence live imaging techniques are also useful in revealing functional, site-specific morphological, and state heterogeneity even at the organelle level in a single cell, which are not observable by simply looking at the cell shape.…”

“…[1][2][3][4]11 However, since organelles are extremely small and difficult to capture within cells, until now, targets that could be captured were limited. We have thus developed methods to capture single vacuoles and single granules.…”

Live Single-cell Metabolomics of Tryptophan and Histidine Metabolites in a Rat Basophil Leukemia Cell

“…We have developed a method involving mass spectrometry (MS) that can directly detect various small molecules in a visualized reacting single-cell, called "video-mass spectrometry" [1][2][3] in which a pico liter level of single-cell content was sucked into a nanospray tip under a microscope and the trapped sample is directly introduced into a mass spectrometer by nano-spray ionization. 1,2 Since the main aim of this new method is to disclose the molecular mechanism of a cell in a direct and rapid way, the analysis of molecular metabolites and those metabolic pathways, i.e.…”

“…1,2 Since the main aim of this new method is to disclose the molecular mechanism of a cell in a direct and rapid way, the analysis of molecular metabolites and those metabolic pathways, i.e. metabolomics, is one of the most important applications.…”