The Development Of Bio-mass Spectrometry Method For Detection Of Ricin In Serum

Ricin is a highly toxic heterodimeric glycoprotein from castor beans (Ricinuscommunis). It is composed of A-chain (RTA) linked to B-chain (RTB) by a singledisulfide bond with a molecular weight of approximately65kDa. RTA is a ribosome-inactivating enzyme that can inhibit protein synthesis. RTB has two galactose bindingsites and can binds to glycoproteins of cell surface and thereby facilitates endocytosicuptake of the ricin. Researchs show that ricin is a potential anti-tumor agent andbiological pesticide, however it can also inhibit protein synthesis and lead to cell deathwhich result in the risk of biological safety and food safety. Ricin was considered to bea biological weapons by the US Centers for Disease Control and Prevention (CDC),which result in the ongoing studies of methods for detection of ricin.There are various methods available for detection of ricin. The most commonmethods are immunological methods based on antibodies which is expensive, labor-intensive and time-consuming (about7hour). Therefore it is necessary to demonstratea rapid and simple method to detect ricin. Two new soft ionization techniques (MALDIand ESI) enables mass spectrometry to detect protein, polypeptide and nucleic acidsand other biological macromolecules with the high sensitivity and wide MS surveyrange, which result in widespread application in the field of life science. Since the1970s,there has been increasing development of magnetic beads for application in separatingand purifying analytes. The magnetic beads can be modified by various chemicalgroups according to characteristics of the analytes and purify analytes from complexmedia with its magnetism. Our preliminary studies have produced Cu-chelatedmagnetic beads and have been successfully applied to the enrichment and purificationof serum peptides, which can be used to judge the curative effect of acute leukemia anddiagnosis of ovarian cancer combining with mass spectrometry. And the method basedon Cu-Magbeads and MALDI-TOF-MS is successfully used for the rapid detection ofpeptide toxins in serum. In this article, we established a method that combines Cu- Magbeads with mass spectrometry for the rapid detection of protein toxin in serum.MALDI-TOF-MS was used to detect the molecular weight of the ricin, andmolecular ion peak (m/z63528Da) was obtained. The disulfide bond between RTA andRTB was reduced by reductant. The mixture was separated by SDS-PAGE and digestedin-gel. The extracted peptides were detected by MALDI-TOF-MS to obtain the peptidemass fingerprinting. Then the ricin solution was subjected to digest using trypsin andchymotrypsin respectively and nanoUPLC-ESI-TOF-MS/MS was used to identify thepeptides. The sequence coverage of ricin was61%after digested by trypsin, while42%after digested by chymotrypsin, and the comprehensive sequence coverage was82%.Structure information of ricin sequence was further confirmed with analysis of peptidecontaining the active site of RTA and C-terminal of RTB. The information above is thebasics of choosing marker peptide after extracting by Cu-Magbeads.In order to improve in-solution protein digestion in terms of speed, an anionicsurfactant (RapiGest) was used, and the digestion time was reduced from12to3hours.We used the Cu-Magbeads coupled with MALDI-TOF-MS to detect ricin and serumafter digestion. By comparing the information, the peptide m/z1728.8Da was selectedto be a marker peptide of ricin and the theoretical sequence was identified to beSAPDPSVITLENSWGR by nanoUPLC-ESI-TOF-MS/MS. For the reproducibility ofthe experiment, we performed six replicate analyses using MALDI-TOF-MS and thecoefficient of variation of the m/z was0.0016%，while S/N was16.10%, indicatingthattherepeatabilityis good.Thelimit ofdetection(LOD)forricin was2ng/μl in waterand5ng/μl in serum. The method was also considered to beapplied to the identificationof tetanus through the experiments.In this study, we established a novel method combining Cu-Magbeads withMALDI-TOF-MS to detect peptide of ricin in serum. Its principle is the histidine,tryptophan and cysteine of protein surface can coordinate with metal ions, and theprotein can be adsorbed and enriched based on the composition of amino acid, locationand different of conformation. This method is rapid, simple, and good reproducibilityto provide a new way for the detection of toxic protein and a base for analysis of toxinwith high flux and sensitivity.