Speaking Plant Approach Combining Pressure Probe and Mass Spectrometry

ERRA-BALSELLS R.; SILVANA L. GIUDICCESI,; YOUSEF GHOLIPOUR; NONAMI H.

Tsukuba, Japón

Conferencia; 58th Annual Conference on Mass Spectrometry Tsukuba; 2010

The Mass Spectrometry Society of Japan

Speaking Plant Approach Combining Pressure Probe and MALDI Mass Spectrometry  (1CIHIDECAR-CONICET, Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, 2Plant Biophysics/Biochemistry Research Laboratory, Faculty of Agriculture, Ehime University, Matsuyama, Japan) Rosa Erra-Balsells1, Hiroshi Nonami2, Yousef Gholipour2 Keywords: tulip bulb, cell pressure probe, UV-MALDI-MS, carbon nanotube Single-cell cytoplasm (1-10 pL) of tulip leaf and bulb cells was extracted by a home-made cell pressure probe equipped with a glass microcapillary tube and micro-manipulator, mixed with 1ìL water in a pipette and deposited on air-dried layer of matrices including 2,5-hydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), nor-harmane (9H-pyrido[3,4-b]indole; nHo), and carbon nanotubes (CNTs) and analyzed with Voyager-DE STR TOF MS (Applied Biosystems) equipped with nitrogen laser (337 nm, 3 ns pulse width) and with 20 kV of accelerating potential. Tissue slices from tulip leaves and bulbs were made by a razor blade and located on the probe, immediately air-dried, and covered by matrices mentioned above and directly analyzed with the MALDI MS after re-drying. The positive ion mode showed superior reproducibility of neutral carbohydrate signals when both single-cell cytoplasm and/or tissues were analyzed. Sucrose and fructans (up to 15Hex when THAP was used, 11Hex with DHB and 7Hex with CNTs) were detected in tulip bulb cell cytoplasm samples. In the case of tissues, only CNTs could efficiently desorb/ionize neutral carbohydrate-related signals from the surface. Hexose, sucrose, and fructan (up to 9Hex) were detected in bulb tissues. Fructans signals were considerably similar in both single-cell and tissue analyses, although, their ionization pattern (as sodium- or potassium- added), was different when using different matrices for cell extract analysiss. In leaf samples, hexoses, sucrose and a triose were detected in both single-cell cytoplasm and tissues. THAP showed to be a candidate matrix for MALDI MS of single cell extract, by which highest m/z of fructans, without decomposition, could be detected. Additionally, analyzing sugar fragments in PSD mode was more convenient when using THAP because of its higher signal reproducibility. In the case of MALDI MS analysis of tissue surface, by which a relative cross-check with single cell extract analysis could be possible, in the lack of capability of organic matrices (i.e. THAP, DHB and nHo) in desorbing/ionizing sugars from the surface of plant tissue, CNTs were the only promising matrix. When using CNTs as matrix deposited on plant tissue, water-lost signal of sugars, probably originated from the partial decomposition of sugars mediated by nanotubes’ surface, was observed. However, characterized carbohydrates and their relative quantity profiled by in situ MALDI MS were similar with those detected by MALDI MS of extracts of single cells as shown in the figure. The method showed to have capability to use for in situ profiling of soluble sugars content of living plant single cells; and profiling tissue surface from which single cell were microsampled for cross-checking.