Quantitative Detection of Trace Systemins in Solanaceous Plants by Immunoaffinity Purification Combined with Liquid Chromatography/Electrospray Quadrupole Time-of-Flight Mass Spectrometry
Quantitative Detection of Trace Systemins in Solanaceous Plants by Immunoaffinity Purification Combined with Liquid Chromatography/Electrospray Quadrupole Time-of-Flight Mass Spectrometry
复制标题
免疫亲和纯化结合液相色谱/电喷雾四极杆飞行时间质谱法定量检测茄科植物中的痕量系统素
DOI：
10.1021/ac101983b
复制
发表时间：
2010-11-15
影响因子：
7.4
通讯作者：
Liu, Huwei
中科院分区：
文献类型：
--
作者：
Du, Fuyou;Bai, Yu;Liu, Huwei
系统素是一类系统性创伤多肽激素，在介导防御害虫攻击和其他创伤中起核心作用。为了更好地了解多肽激素的分子机制，需要建立一种灵敏、准确的检测方法来监测植物中痕量的系统素。建立了一种基于免疫亲和柱（IAC）纯化富集、液相色谱-电喷雾四极杆飞行时间质谱（LC/ESI QTOF MS）在线联用的茄科植物中微量系统素的上级鉴定和定量分析方法。制备了番茄系统素多克隆抗体，并将其固定在溴化氰活化的Sepharose固定相上。利用所制备的IAC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 H）、辣椒系统素（PepSys）和茄属植物系统素（NishSys）。随后通过LC/ESI QTOF MS进行的鉴定和测定表明，IAC能够有效和特异性地富集PotSys I，PotSys II，尤其是TomSys。在优化的条件下，该方法成功地应用于番茄叶片中TomSys和马铃薯叶片中PotSys I和PotSys II的测定，并提供了检测限（LOD，S/N =3）为29、72和135 pg/g，三种浓度下的平均回收率分别为92.9%、56.7%和34.8%（TomSys、PotSys I和PotSys II分别为1.0、2.0和4.0 ng/g）。
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 H), 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.