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Innovations Forum: Proteome analysis of A. thaliana Proteome analysis of Arabidopsis thaliana mitochondrial proteins L. Sweetlove*, J. Flensburg†, O. Rönn†, H.-O. Gustavsson†, and E. Forsberg† *Department of Plant Sciences, University of Oxford, Oxford, UK; †Amersham Biosciences AB, Uppsala, Sweden Mitochondria were isolated from Arabidopsis thaliana cells and the cell extracts analyzed by 2-D electrophoresis-mass spectrometry (2D-MS). Low-abundance proteins were analyzed by peptide-mass fingerprinting (PMF) using Ettan™ MALDI-ToF Pro. Peptide-mass fingerprinting of 50 selected spots from the 2-D electrophoresis gel resulted in a 74% protein identification rate. Proteins not successfully identified by PMF were analyzed by chemically assisted fragmentation MALDI using Ettan CAF™ MALDI Sequencing Kit in combination with Ettan MALDI-ToF Pro. Using this approach increased the identification rate of the selected mitochondrial proteins to 86%. Introduction During the last decade, Arabidopsis thaliana, a small flowering weed, has developed into a model system for plant development, physiology, and genetics. Scientists all over the world are using new tools to investigate the plant’s proteome to discover the processes common to all plants. A. thaliana is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish. The sequencing of the whole genome of A. thaliana was completed during 2000 by the international Arabidopsis Genome Initiative (1). In this study, an analysis of the mitochondrial proteome was undertaken. Plant mitochondria have a multiplicity of functions other than that of ATP production. Many of these, such as synthesis of ascorbic acid and oxidation of the amino acids proline and glycine, are unique to plants. However, the identity of the complete set of mitochondrial proteins that constitute the mitochondrial proteome has not been resolved. Hence, the complete set of metabolic functions of plant mitochondria remains undefined. Some 95% of the genes encoding mitochondrial proteins are located in the nucleus and the proteins are targeted back to the mitochondrion using encrypted targeting information in the protein sequence. Identification of all genes carrying these targeting sequences is one way of defining the complete set of mitochondrial proteins. However, the presence of multiple types of targeting sequences, and the lack of homology between consensus targeting sequences in plants and animals, diminish confidence in this approach. The genome of A. thaliana has been analyzed in this fashion and the number of gene products with a predicted mitochondrial location range from as little as 349 to as many as 2897 (1). Since the initial launch of the complete genome sequence of A. thaliana, however, attempts have been made to improve the accuracy of gene models. Introduction of improved gene prediction algorithms as well as annotation based on increased availability of full-length cDNAs has dramatically improved the accuracy of the A.thaliana genome information (2). To ascertain whether this improves the ability to identify proteins by PMF, we repeated the analysis of some A. thaliana mitochondrial proteins, described earlier in reference 3. A total of 50 proteins were analyzed using twodimensional electrophoresis-mass spectrometry (2D-MS). Peptide-mass fingerprinting using Ettan MALDI-ToF Pro was used for the first tier of protein identification. To further improve the protein identification rate using MALDI-ToF, chemically assisted fragmentation MALDI was used to analyze proteins that were not successfully identified by PMF. This technique enables peptide sequence data to be acquired simply, quickly, and with good sensitivity using MALDI ToF MS. Materials and Methods First-dimension electrophoresis was performed using Immobiline™ DryStrip pH 3–10 NL, 18 cm on Multiphor™ II IEF System. Seconddimension separation was performed using Ettan DALTtwelve Large Vertical Electrophoresis System using large format, lab-cast SDS gels. The gel was stained with colloidal Coomassie™ Brilliant Blue (American Bioanalytical) and fifty spots of low staining intensities were selected for further manipulation. Life Science News 15, 2003 Amersham Biosciences 13 Innovations Forum: Proteome analysis of A. thaliana Using Ettan Spot Handling Workstation, selected spots were automatically picked, tryptically digested, and the resultant tryptic peptides co-spotted with α-cyano-4-hydroxycinnamic acid matrix solution onto the MALDI sample slide. In the current analysis, identification of these six proteins was confirmed and the eight previously unidentified proteins were this time successfully identified (Table 1). This clearly demonstrates that Protein identification was performed by PMF using the Ettan MALDI-ToF Pro mass spectrometer. The entire process for protein identification by PMF was fully automated from data acquisition, through calibration, spectral processing, and subsequent databsase searching. Proteins not successfully identified by PMF were further analyzed by chemically assisted fragmentation MALDI using Ettan CAF MALDI Sequencing Kit in combination with Ettan MALDI-ToF Pro. The relevant peptide digests were derivatized using the kit according to the manufacturer’s instructions. Results and discussion We were able to consistently resolve approximately 100 highabundance proteins and 250 low-abundance proteins in an experimental setup using a wide pH 3–10 nonlinear gradient on the gels in the first dimension (Fig 1). Fifty of these proteins, most of them representing low-abundance proteins on the 2-D electrophoresis gel, were analyzed. After automated spot handling, PMF identified 74% of these proteins (37 of 50 [Fig 2]). Fourteen of the 50 proteins in this study had been analyzed earlier (3), where only six proteins (43%) were successfully identified. Fig 2. Peptide-mass fingerprinting of selected mitochondrial proteins yielded an unambiguous match from an enquiry of the A. thaliana database. improvements in genomic database annotations, as well as the superior mass accuracy of Ettan MALDI-ToF Pro contribute to a very high success rate of protein identification through PMF. The remaining unidentified samples were analyzed by chemically assisted fragmentation MALDI. The CAF reagent included in Ettan CAF MALDI Sequencing Kit, and initially reported by Keough and colleagues in 1999 (4), allows the rapid and efficient acquisition of peptide sequence information using MALDI-ToF. Chemically assisted fragmentation MALDI offers two key benefits. One is that fragmentation efficiency of singly charged peptides produced during MALDI post-source decay is enhanced, thereby increasing sensitivity. The other is that only a single fragment ion series (y-ion series) is observed, thereby increasing sensitivity and simplifying the spectrum (applicable to any MALDI MS/MS spectrum). In this study, six of the 13 proteins not identified by PMF were unambiguously identified by chemically assisted fragmentation MALDI using Ettan CAF MALDI Sequencing Kit. In the example shown in Figure 4, nine consecutive residues of sequence were obtained and used to identify the protein. Using this technique, the protein identification rates from a single MALDI were increased to 86%. Ettan CAF MALDI Sequencing Kit is equally applicable to any MS instrument equipped with a MALDI ionization source. Fig 1. Two-dimensional electrophoresis of the A. thaliana mitochondrial proteome. Firstdimension separation: 18 cm Immobiline DryStrip pH 3–10 NL IPG strips, Ettan IPGphor™ IEF System. Second-dimension separation: Large-format lab-cast gels, Ettan DALTtwelve Large Vertical Electrophoresis System. Staining: Coomassie™ Brilliant Blue. 14 Life Science News 15, 2003 Amersham Biosciences Innovations Forum: Proteome analysis of A. thaliana OCH3 A C NH N2N CH NH CO CH NH O-Methylisourea-hydrogen sulfate CH2 CO CH2 CH2 CH2 CH2 CH2 CH2 CH2 NH2 NH2 lysine C H2N NH homoarginine (+42 amu) O O N O H O R1 S OH + N2H R2 O O O Pep OH S R1 O O B Conclusions H NH Fig 4. Chemically assisted fragmentation MALDI spectrum. Nine consecutive residues of sequence were acquired, which allowed successful identification of mitochondrial proteins in the A. thaliana database. Pep R2 sulfonated N-terminal (+136 amu) Fig 3. Reaction scheme for the chemically assisted fragmentation derivatization step of Ettan CAF MALDI Sequencing Kit. (A) The first step is protection of the ε-amino group of the lysine side chain. (B) The second step is sulfonation of the α-amino N-terminus of the peptide. The 2-D MS workflow is a valuable tool to characterize a plant mitochondrial proteome, enabling low-abundance proteins to be detected and identified. Automated PMF analysis gave an identification rate of 74% (37 proteins of 50 total). The automated software of Ettan MALDI-ToF Pro enables protein identification with minimal manual intervention. Chemically assisted fragmentation MALDI, which is a relatively simple technique, further improved protein identification rates to 86%. Reference Table 1. Improved protein identification of A. thaliana mitochondrial proteins using Ettan MALDI-ToF Pro Spot number Protein identity (see reference 2) Protein identity (Ettan MALDI-ToF Pro) 1. Arabidopsis Genome Initiative-Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815 (2000). 2. Wortman, J. R., et al. Annotation of the Arabidopsis genome. Plant Physiol 132, 461–468 (2003). 3. Millar, A. H., et al. Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127, 1711–1727 (2001). 1 glycine-rich RNA-binding protein glycine-rich RNA-binding protein 2 manganese superoxide dismutase (MSD1) manganese superoxide dismutase (MSD1) 3 ATP-dependent Clp protease proteolytic subunit (ClpP2), putative ATP-dependent Clp protease proteolytic subunit (ClpP2), putative 4 glutathione transferase, putative glutathione transferase, putative 5 glutathione transferase, putative glutathione transferase, putative 6 glutathione transferase, putative glutathione transferase, putative Ordering Information 7 not identified 10 kDa chaperonin (CPN10) Multiphor II IEF System 18-1018-06 8 not identified hypothetical protein Immobiline DryStrip pH 3–10 NL, 18 cm 17-1235-01 9 not identified F1F0-ATPase inhibitor protein 80-6466-46 10 not identified armadillo repeat containing protein Ettan DALTtwelve Large Vertical System, 115 V 11 not identified expressed protein Ettan DALTtwelve Large Vertical System, 220 V 80-6466-27 12 not identified unknown protein Ettan CAF MALDI Sequencing Kit 17-6002-97 13 not identified plastid protein –related 18-1156-54 14 not identified expressed protein Ettan MALDI-ToF Pro Mass Spectrometer, 120 VAC Ettan MALDI-ToF Pro Mass Spectrometer, 240 VAC 18-1156-53 Ettan Spot Handling Workstation 18-1164-05 4. Keough, T., et. al. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. Proc. Natl. Acad. Sci. USA 96, 7131–7136 (1999). To obtain a brochure on Ettan MALDI-ToF Pro, please circle 3 on the reader reply card, or download it at www.lsn-online.com/info. Life Science News 15, 2003 Amersham Biosciences 15