Highly sensitive MALDI-MS measurement of active ricin: insight from more potential deoxynucleobase-hybrid oligonucleotide substrates
Herein, we report an improved MALDI-MS method for active ricin to contribute toward countermeasures against its real threat to the public. Compared with commonly used DNA or RNA substrates, the deoxynucleobase-hybrid oligonucleotide (RNA_dA, Rd) substrate containing functional GGA loop was revealed as a substrate with more potential and used for the first time in ricin measurement via MALDI-MS. The Rd sequence greatly prompted ricin to exhibit its catalytic activity as rRNA N-glycosylase in ex vitro condition, which was supported by molecular docking simulation and enzymatic parameters depicted in MALDI-MS. Furthermore, we discovered that a highly pure matrix was the most crucial parameter for enhancing the sensitivity, which addressed the major obstacle encountered in the oligo(deoxy)nucleotide measurement, i.e., the interfering alkali metal ion-adducted signals in MALDI-MS. After the optimization of pH and enzymatic reaction buffer composition in this ex vitro condition, this method can provide a wide linearity of up to three orders of magnitude, i.e., 1–5000 ng mL−1, and a high sensitivity of 1 ng mL−1 without any enrichment. Denatured and active ricin could be distinctly differentiated, and the application to practical samples from one international exercise and a soft drink proved the feasibility of this new method. We believe this MALDI-MS method can contribute to the first response to ricin occurrence events in public safety and security, as well as pave a new way for a deep understanding of ricin and other type II ribosome inactivating proteins involved toxicology.
危险品化学品经营许可证（带存储）