In Situ Pressure Probe Picoliter Single-Cell Sap Sampling and Mass Spectrometry Metabolite Profiling of Living Plants.

Y. GHOLIPOUR; ERRA-BALSELLS, ROSA; H. NONAMI; ERRA BALSELLS, ROSA

Kyoto

Conferencia; 19th International Mass Spectrometry Conference, IMSC2012 Kyoto,; 2012

International Mass Spectrometry Foundation

In Situ Pressure Probe Picoliter Single-Cell Sap Sampling and Mass Spectrometry Metabolite Profiling of Living Plants Yousef Gholipour,1 Rosa Erra-Balsells2 and  Hiroshi Nonami1 1Plant Biophysics/Biochemistry Research Laboratory, Faculty of Agriculture, Ehime University, Matsuyama, Japan 2 CIHIDECAR-CONICET, Departamento de QuimicaOrganica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina Key words: Intact cell, quantitation, metabolomics, glycomics, Novel aspects: Mass spectrometry analysis of picoliter single-cell sap sampled by using the pressure probe is performed. Single-cell sap volume sucked is controlled and measured. Additional intact-cell properties can be evaluated. In plants, anatomy of different organs and tissues is not necessarily uniform and cells located at different tissues and depth may vary significantly in function, morphology and metabolite composition. Therefore, single-cell metabolite profiling to be applied to plant science demands for accurate and fully-managed sampling from different tissues and cell layers. The sampling is more challenging due to the difficulty in managing femto- to nanoliter volume of sap each plant cell can provide. Additionally, the single-cell sap volume must be measured if natural changes in the abundance of metabolites of cells are to be analyzed. In our model plant, tulip, parenchyma cells located at inner layers of bulb scale tissue (e.g. 400 ìm from the surface of cuticle) contain abundant starch, long-chained fructans, underivatized soluble sugars and amino acids.  They function as the source of nitrogen and carbon for growth of new flower stalk. For flowering, bulbs need to be stored at low temperatures (4-10°C) for about two months. We targeted those parenchyma cells in cold-stored bulbs.. We used a cell pressure probe for in situ single-cell sampling followed by shotgun metabolite profiling with UV-MALDI TOF MS (AB SCIEX 5800 system) and nanoESI MS (ThermoFisher Exactive Orbitrap? mass spectrometer). The quartz capillary of the pressure probe was filled with silicon oil and air-tightly connected to a pressure transducer. Pressure inside capillary is manipulated with a rod moved back and forth by a micrometer. Therefore, while the capillary was penetrating to a pre-determined tissue depth, the sap of cells located on the way to the target cell was prevented from entering the capillary tip by regulating the oil pressure. The movement of capillary and penetration depth was controlled and measured with a piezo-manipulator and with previous anatomy of tulip bulb tissues, depth of penetration to access favorite cells became possible. After penetration of the capillary into a cell, cell sap enters the capillary tip due to turgor pressure naturally exists in living plant cells; and a meniscus forms at the interface of the oil and analyte solution inside the capillary. The diameter of parenchyma cells in tulip bulb scale varies in the range of 50-150 um and we obtained 100-600 pL cell sap from each cell. The tip with cell sap sample inside was photographed and the volume of the picoliter sample was accurately measured. For UV-MALDI MS, the picoliter cell sample was injected to a 1 ìL water droplet hanging from a pipette tip. The picoliter cell sample (in 1 ìL water droplet) was deposited on previously dried matrix spots on stainless steel MALDI plates. We have examined several matrixes for single-cell metabolite profiling and in this study we used 2,4,6-trihydroxyacetophenone (THAP), 2,5-dihydroxybenzoic acid (DHB) and titanium silicon oxide (SiO2)(TiO2) nanoparticles. With (SiO2)(TiO2) nanoparticles good linearity of signal abundance vs. number of picomoles of standard compounds was yielded. With all matrixes, signals of cell sap metabolites were detected. Particularly, big neutral oligosaccharides could be easily analyzed with UV-MALDI MS. For nanoESI MS analyses, cell sap sample was transferred to a 5 ìL water droplet and then injected into the electrospray ion source. NanoESI MS was also successful in metabolite profiling of single-cell sample; however, smaller oligosaccharides could be detected. In addition to sugars, amino acids, organic acids, tuliposide, putrescine, and GABA could be detected with both MALDI and nanoESI MS. Relative abundance of sucrose and kestose in parenchyma cells located at the depth of 100-500 ìm was determined. The limit of detection of sucrose was similar (2-5 pmol) in both techniques but dynamic range of detection was wider in MALDI MS (with 2-25000 pmol) than nanoESI MS (5-2500 pmol).