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.
Anal Chem. 2010 Nov 15;82(22):9374-83. doi: 10.1021/ac101983b. Epub 2010 Oct 20.
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.
系统素是一类全身性伤口多肽激素，在介导防御害虫攻击和其他伤害方面发挥着核心作用。人们一直希望开发一种敏感而准确的测定方法来监测植物中的痕量系统素，以更好地了解多肽激素的分子机制。本工作描述了一种在茄科植物中准确鉴定和定量测定痕量系统素的优越方法，该方法基于免疫亲和柱（IAC）纯化和富集，然后与液相色谱在线耦合电喷雾四极杆飞行时间质谱（LC/ESI QTOF MS）。已经制备了特异性抗番茄系统素多克隆抗体并将其固定在 CNBr 活化的琼脂糖固定相上。制备的 IAC 用于从茄科植物中提取和富集番茄系统素（TomSys）、马铃薯系统素（PotSys I 和 PotSys II）、辣椒系统素（PepSys）和茄属植物系统素（NishSys）。通过 LC/ESI QTOF MS 进行后续鉴定和测定表明，IAC 能够有效地特异性富集 PotSys I、PotSys II，尤其是 TomSys。在优化条件下，该方法成功应用于番茄叶片中 TomSys、马铃薯叶片中 PotSys I 和 PotSys II 的测定，其检测限（LOD，S/N=3）分别为 29、72 和 135 pg/g，TomSys、PotSys I 和 PotSys II 的平均回收率分别为 92.9%、56.7%和 34.8%，在三个浓度（1.0、2.0 和 4.0 ng/g）下。
