Quantitative Detection of Trace Systemins in Solanaceous Plants by Immunoaffinity Purification Combined with Liquid Chromatography/Electrospray Quadrupole Time-of-Flight Mass Spectrometry

Abstract

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.