Cell-specific protein profiling in Arabidopsis thaliana trichomes: identification of trichome-located proteins involved in sulfur metabolism and detoxification Stefanie Wienkoop, Daniela Zoeller, Berit Ebert, Ulrike Simon-Rosin, Joachim Fisahn, Mirko Glinski, Wolfram Weckwerth * Max Planck Institute of Molecular Plant Physiology, Metabolic Networks, 14424 Potsdam, Germany Received 8 January 2004; received in revised form 23 March 2004 Available online 1 June 2004 Abstract Metabolite, protein, and transcript analysis at the cellular level gives unparalleled insight into the complex roles tissues play in the plant system. However, while capillary electrophoresis and PCR amplification strategies make the profiling of metabolites and transcripts in specific cell types possible, the profiling of proteins in small samples represents a bottleneck. Here for the first time protein profiling has been achieved in a specific plant cell type: The application of specific cell sampling and shotgun peptide se- quencing (nano LC/MS/MS) resulted in the identification of 63 unique proteins from pooled Arabidopsis trichome cells. A complete S-adenosylmethionine pathway cluster, two S-adenosylmethionine synthase isoforms, a glutathione S-conjugate translocator and other proteins involved in sulfur metabolism and detoxification are shown to be present in these cells, in agreement with previous work done at the level of trichome transcript analysis. The technology described here brings the simultaneous identification and localization of physiologically relevant cellular proteins within reach. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Plant shotgun proteomics; Nano-LC/MS; Sulfur metabolism; Single cells 1. Introduction High spatial and temporal resolution of transcript and metabolite profiling at the single-cell level requires extremely sensitive analytical methods: capillary elec- trophoresis and PCR amplification strategies combined with specific sampling methods have been used suc- cessfully at this level (Fricke et al., 1994; Brandt et al., 1999, 2002; Tomos and Leigh, 1999; Koroleva et al., 2000; Kehr, 2001; Lochmann et al., 2001; Nakazono et al., 2003). Initial studies on single cell proteins using two-dimensional gel electrophoresis (2DE) in combina- tion with native gels and mass spectrometry allowed the analysis of target proteins from guard cells (Li and Assmann, 2000). However, until now large-scale iden- tification of proteins on the cell-specific level of plants failed due to small sample volumes, low protein levels, and complex tissue structure (Kehr, 2001, 2003). The use of oligonucleotide- and cDNA-based microarrays is providing unprecedented insights into the regulation of global patterns of gene expression (Schulze and Down- ward, 2001). Nevertheless, since protein abundance does not always correlate with transcript levels (Gygi et al., 1999a; Greenbaum et al., 2003) and since the cellular localization and turnover rate of biologically active protein can only be determined directly, the develop- ment of sensitive methods for comprehensive analysis of specific cell type protein expression patterns in Arabid- opsis and other plants is needed. The combination of HPLC separation of complex peptide mixtures with subsequent tandem mass spectrometry (LC/MS/MS) provides an alternative tool for proteome and subpro- teome analysis (Link et al., 1999; Yates et al., 1999; * Corresponding author. Tel.: +49-331-567-8109; fax: +49-331-567- 8134. E-mail address: weckwerth@mpimp-golm.mpg.de (W. Weckwerth). 0031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.03.026 Phytochemistry 65 (2004) 1641–1649 PHYTOCHEMISTRY www.elsevier.com/locate/phytochem