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Quantitative Detection of Trace Systemins in Solanaceous Plants by Immunoaffinity Purification Combined with Liquid Chromatography/ Electrospray Quadrupole Time-of-Flight Mass Spectrometry Fuyou Du, Yu Bai, Yu Bai, and Huwei Liu* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Systemins are a class of systemic wound polypeptide hormones that play a central role in mediating defenses against pest attacks and other woundings. It has been desired to develop a sensitive and accurate determination method to monitor trace systemins in plants for the better understanding of molecular mechanisms of the polypeptide hormones. A superior method for accurate identification and quantitative determination of trace systemins in Solanaceous plants is described in this work, which is based on immunoaffinity column (IAC) purification and enrichment followed by liquid chromatography online coupled to electrospray quadrupole time-of-flight mass spectrometry (LC/ESI QTOF MS). The specific antitomato systemin polyclonal antibody had been produced and immobilized on a CNBr-activated Sepharose stationary phase. The prepared IAC was utilized for the extraction and enrichment of tomato systemin (TomSys), potato systemin (PotSys I and PotSys II), pepper systemin (PepSys), and nightshade systemin (NishSys) from Solanaceous plants. Subsequent identification and determination by LC/ESI QTOF MS revealed that the IAC enables efficient and specific enrichment of PotSys I, PotSys II, and especially TomSys. Under the optimized conditions, the developed method was successfully applied in the determination of TomSys in tomato leaves and PotSys I and PotSys II in potato leaves, and it offers detection limits (LODs, S/N )3) of 29, 72, and 135 pg/g and mean recoveries of 92.9%, 56.7%, and 34.8% at three concentrations (1.0, 2.0, and 4.0 ng/g) for TomSys, PotSys I, and PotSys II, respectively. Systemin, an 18-amino-acid wound polypeptide hormone, was first isolated in 1991 by Ryan et al.1 from infected leaves of the tomato Lycopersicon esculentum. It activates and regulates the synthesis of antinutritive proteins such as proteinase inhibitors, polyphenol oxidase, polygalacturonase, and amino acid catabo* Corresponding author. Phone: +86-10-62754976. Fax: +86-10-62751708. E-mail:
[email protected]. (1) Pearce, G.; Strydom, D.; Johnson, S.; Ryan, C. A. Science 1991, 253, 895– 897.
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lizing enzymes not only in the damaged tissues but also in the distant undamaged parts of the plant2-8 and systemically regulates over 20 defensive genes in tomato plants in response to herbivore and pathogen attacks and other mechanical woundings.9 The characteristics of systemin, including its potency, its mobility in the plant, the wound-inducibility of the prosystemin gene, the effects of the antisense gene in blocking systemic wound signaling, and the reestablishment of the systemic wound response in antisense plants have led to the conclusion that systemin is a systemic wound signal hormone and plays a key role in the regulation of plant defense, reproduction, growth, and development.9-19 Besides tomato systemin (TomSys), other systemins have also been isolated and identified in other plant species of the Solanaceae family, including potato (Solanum tuberosum), bell pepper (Capsicum annuum), and black nightshade (Solanum nigrum), and their peptide sequences differ slightly from each other (Table (2) Ryan, C. A.; Pearce, G.; Johnson, S.; McGurl, B.; Orozco-Cardenas, M.; Farmer, E. E. Plant Pathol. 1992, 2, 196–201. (3) McGurl, B.; Ryan, C. A. Plant Mol. Biol. 1992, 20, 405–409. (4) McGurl, B.; Orozco-Cardenas, M. L.; Pearce, G.; Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 9799–9802. (5) Narvaez-Vasquez, J.; Orozco-Cardenas, M. L.; Ryan, C. A. Plant Physiol. 1994, 105, 725–730. (6) Constabel, C. P.; Bergey, D. R.; Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 407–411. (7) Bergey, D. R.; Orozco-Cardenas, M.; Demoura, D. S.; Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1756–1760. (8) Chen, H.; Wilkerson, C. G.; Kuchar, J. A.; Phinney, B. S.; Howe, G. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 19237–19242. (9) Ryan, C. A. Biochim. Biophys. Acta 2000, 1477, 112–121. (10) Schaller, A.; Ryan, C. A. BioEssays 1995, 18, 27–33. (11) Bergey, D. R.; Howe, G. A.; Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12053–12058. (12) Schaller, A. Plant Mol. Biol. 1999, 40, 763–769. (13) Lindsey, K.; Casson, S.; Chilley, P. Trends Plant Sci. 2002, 7 (2), 78–83. (14) Ryan, C. A.; Pearce, G.; Scheer, J.; Moura, D. S. Plant Cell 2002, S251– S264. (15) Ryan, C. A.; Moura, D. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6519– 6520. (16) Ryan, C. A.; Pearce, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14577– 14580. (17) Stratmann, J. W. Trends Plant Sci. 2003, 8, 247–250. (18) Schilmiller, A. L.; Howe, G. A. Curr. Opin. Plant Biol. 2005, 8, 369–377. (19) Kandoth, P. K.; Ranf, S.; Pancholi, S. S.; Jayanty, S.; Walla, M. D.; Miller, W.; Howe, G. A.; Lincoln, D. E.; Stratmann, J. W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12205–12210. 10.1021/ac101983b 2010 American Chemical Society Published on Web 10/20/2010
Table 1. Results of the Cross-Reactivities of TomSys with Other Systemins (n ) 4) peptide (abbreviation)
amino acid sequence
cross-reactivity (%)
tomato systemin (TomSys) potato systemin I (PotSys I) potato systemin II (PotSys II) nightshade systemin (NishSys) pepper systemin (PepSys) tobacco systemin (TobHypSys I)
AVQSKPPSKRDPPKMQTD AVHSTPPSKRDPPKMQTD AAHSTPPSKRDPPKMQTD AVRSTPPPKRDPPKMQTD AVHSTPPSKRPPPKMQTD RGANLPOOSOASSOOSKE
100 57 35 26 23
