Pressure probe electrospray ionization for one cell metabolite/protein analyses.

T. NAKASHIMA, ; H. WADA, ; S. MORITA, ; A. TAKEMORI, .; R. ERRA-BALSELLS, ; K. HIRAOKA, ; H. NONAMI

Tsukuba

Conferencia; The 63th Annual Conference on Mass Spectrometry, Tsukuba, Japan, 17-19 Junio 2015; 2015

The Mass Spectrometry Society of Japan

Pressure probe electrospray ionizationfor one cell metabolite/protein analyses (Faculty of Agriculture, EhimeUniversity1, Kyushu Okinawa Agricultural Research Center2,Proteo-Science Center, Ehime University3, Clean Energy Research Center,University of Yamanashi4, University of Buenos Aires5) TaikenNakashima1, Hiroshi Wada2, Satoshi Morita2,Nobuaki Takemori3, Ayako Takemori3, Kenzo Hiraoka5,Hiroshi Nonami1,3, ??Rosa Erra-Balsells4Keywords: Pressure probe, Single cell, Metabolomics, Proteomics,Plant cellsDirectly sampled cell solution can be ionized for MSanalysis by applying high voltage to the solution inside a tip of the capillaryof the pressure probe containing silicon oil mixed with a small amount of ionicliquid. Trichomes are unicellular or multicellular outgrowths originating fromthe aerial epidermal cells of stems, leaves and floral organs and fruits. In Solanum spp., they are typicallyclassified into eight distinct types based on their morphological appearance.Some types of trichomes are known to synthesize and accumulate specializedmetabolites such as acylsugars, flavonoids and terpenoids. Their contents mayvary transiently through developmental stages of plants. Monitoring thephysicochemical and metabolic properties of individual trichome cells is,therefore, a potential means to provide diagnostic clues for physiologicalstatus of crop plants. Metabolite profiling in intact single cells fromdifferent types of trichomes in tomato plants is shown by using a pressureprobe electrospray ionization mass spectrometry (PPESI-MS) system. Proteins intomato trichomes can be also analyzed in PPESI-MS. Additionally, real timepressure probe operation is shown in rice grains for rice chalky ring formationcaused by temporal reduction in starch biosynthesis during osmotic adjustmentunder foehn-induced dry wind1).The cell pressure probe was originally designed toassess the water status and several physical properties of intact plant cells byimpaling a finely tapered quartz microcapillary1), 2). After the pressureprobe operation, cell sap trapped in the capillary tip can be directly ionizedby applying high voltage to the tip, and ionized cellular components are detectedby high-resolution Orbitrap MS. We previously reported successful detection andidentification of a number of metabolites from parenchyma cells of tulip bulbsusing PPESI-MS3). The reliability of this technique has beenconfirmed by complementary nanoESI analysis of diluted cell samples. Also,MALDI and PESI MS analyses gave the similar results in the similar tulip cellsamples2), 3).Recently, a series of modifications to the system ledto drastic increases in throughput capacity and detection sensitivity of somestandard metabolites as well as peptides, allowing us to perform an efficientand highly-sensitive metabolite profiling of intact tomato trichomes at singlecell resolution. The minimum sample volume required for metabolite analysis ofa single stalk cell was as low as sub-pL. In addition, the capability of the pressureprobe for precise post-sampling manipulation enabled dilution and subsequent ionizationof highly viscous component of glandular cells of tomato plants. Consequently,we were able to characterize remarkable variations in metabolite profiles betweenneighboring stalk and glandular cells in the same trichome type and amongdifferent trichome types. The application of this technique was further extendedto proteomics analysis of trichomes. AcknowledgementsThis work was supported in part by JSPS KAKENHI (Grant-in-AidScientific Research) (S), (A) and (B). ReferencesWada, H.,Masumoto-Kubo, C., Gholipour, Y., Nonami, H., Tanaka, F., Erra-Balsells, R.,Tsutsumi, K., Hiraoka, K., Morita, S. (2014) PLoS ONE 9(10): e110374.doi:10.1371/journal.pone.0110374Gholipour, Y., Erra-Balsells,R., Nonami, H. (2012) Mass Spectrometry 1: DOI: 10.5702/massspectrometry.A0003Gholipour, Y.,Erra-Balsells, R., Hiraoka, K., Nonami, H. (2013) Anal. Biochem. 433:70-78,doi: 10.1016/j.ab.2012.10.001