NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Neoadjuvant Chemotherapy or Adjuvant Chemotherapy for Esophageal Squamous Cell Carcinoma.

BACKGROUND: Chemotherapy and chemoradiation have become essential adjuncts to improve the survival of patients with resectable esophageal squamous cell carcinoma (ESCC) in the perioperative period. Although preoperative treatment plus surgery is commonly used, controversy remains regarding the optimal treatment strategy for patients with locally advanced ESCC. METHODS: A retrospective review of clinical stage II and III ESCC patients who underwent esophagectomy at Henan Cancer Hospital between October 2014 and October 2017 was performed. The patients were divided into a neoadjuvant chemotherapy (NAC) group and an adjuvant chemotherapy (AC) group. Propensity score matching (PSM) was used to exclude confounders. Survival was estimated using Kaplan‒Meier analysis and compared by the log-rank test. The Cox proportional hazards regression model was used for both the univariate and multivariate analyses. RESULTS: A total of 684 patients were enrolled, including 365 (53.4%) patients in the NAC group. After PSM, 294 pairs of patients were left. NAC prolonged the OS (not reached versus 57.3 months, P = 0.002) and DFS (57.2 vs. 36.4 months, P = 0.010) and decreased the total rate of recurrence (50.1% vs. 59.2%, P = 0.025) and local recurrence (27.9% vs. 36.7%, P = 0.022) compared with AC. The multivariable analyses showed that NAC plus surgery modality was an independent predictor for improved OS (HR: 0.582, 95% CI: 0.467-0.786, P = 0.001). CONCLUSION: NAC plus surgery prolonged OS and DFS, and significantly decreased the total rate of recurrence compared with surgery plus AC in patients with clinical stage II and III ESCC.

Simultaneous Quantification of Biomarkers for Bis-(2-chloroethyl) Sulfide and 1,2-Bis(2-chloroethylthio) Ethane Exposure in Human Urine at Trace Exposure Levels by Gas Chromatography Tandem Mass Spectrometry via Simultaneous Incubation and Extraction.

Sulfur mustard [HD; bis-(2-chloroethyl) sulfide] and other analogues are a kind of highly toxic vesicant and have been prohibited by the Organization for the Prohibition of Chemical Weapons (OPCW) since 1997. Exposures to HD could generate several adducts in the plasma and hydrolysis products in the urine, which are widely applied as biomarkers to identify HD exposure in forensic analysis. Several methods have been developed for the detection of related biomarkers. However, most methods are based on complex derivatization, and not enough attention is paid to HD analogues. A modified and convenient analytical method reported herein includes simultaneous incubation and organic solvent extraction. The biomarkers such as thiodiglycol and 1,2-bis (2-hydroxyethylthio) are transferred to HD and 1,2-bis(2-chloroethylthio) ethane via hydrochloric acid at the appropriate temperature. The analytes are analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) with 2-chloroethyl ethyl sulfide (2-CEES) applied as the internal standard. The interday and intraday study according to FDA rules has been achieved to evaluate the accuracy and precision of the method. The two targets are detected with a good linearity (R2 > 0.99) in the concentration ranges from 5 to 1000 ng/mL and 10 to 1000 ng/mL, with small relative standard deviations (RSD ≤6.62% and RSD ≤6.93%) and favorable recoveries between 90.3 and 107.3% and between 89.4 and 108.7%, respectively. The established method can be used for retrospective detection of sulfur mustards in biological samples and successfully applied in the biomedical proficiency testing organized by the OPCW.

A multicenter randomized, controlled clinical trial of adjuvant sintilimab for esophageal squamous cell carcinoma.

No adjuvant treatment has been established for patients who remain at high risk of recurrence and incidental pathologic lymph node metastasis for esophageal squamous cell carcinoma (ESCC). In this open-label, multicenter, phase III, randomized controlled trial, ESCC patients who did not achieve pathologic complete response after neoadjuvant chemotherapy plus surgery and clinical T1-2 N0 patients with incidental pathologic lymph node metastasis following initial surgery were randomized at a 2:1 ratio to receive either a sintilimab regimen or observational management (NCT05495152). The primary end point was disease-free survival for all randomized patients. The results of this randomized controlled trial addressed controversy regarding the survival benefits of adjuvant sintilimab treatment for patients with resected locally advanced ESCC. Clinical Trial Registration: NCT05495152 (ClinicalTrials.gov).

Ponicidin inhibited gallbladder cancer proliferation and metastasis by decreasing MAGEB2 expression through FOXO4.

BACKGROUND: Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC. METHODS: CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin. RESULTS: Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety. CONCLUSION: Ponicidin may be a promising agent for the treatment of GBC effectively and safely.

Initial experience with modified en bloc robot-assisted minimally invasive oesophagectomy for thoracic oesophageal squamous cell carcinoma.

BACKGROUND: The feasibility and safety of en bloc robot-assisted minimally invasive oesophagectomy (RAMIE) need to be verified. METHODS: Forty-seven patients who received conventional RAMIE and 31 who received modified en bloc RAMIE at Henan Cancer Hospital were included in the study cohort. We compared the perioperative outcomes of conventional RAMIE and modified en bloc RAMIE. RESULTS: Compared with the conventional RAMIE group, the en bloc RAMIE group yielded a higher total number of lymph nodes (p = 0.001), especially thoracic lymph nodes (p = 0.025) and left recurrent laryngeal nerve (RLN) lymph nodes (p = 0.005). No notable differences were found in the rate of total complications (p = 0.663) or RLN injury (p = 0.891) between the two groups. The preoperative and postoperative serological indicators were comparable between the two groups. CONCLUSIONS: Modified en bloc RAMIE was safe and feasible for patients with oesophageal squamous cell carcinoma and improved lymph node dissection, especially thoracic and left RLN lymph node dissection.

Inhibition of XPO1 impairs cholangiocarcinoma cell proliferation by triggering p53 intranuclear accumulation.

BACKGROUND: XPO1 mediates the nuclear export of several proteins, mainly tumor suppressors. KPT-330 (Selinexor) is a selective inhibitor of XPO1 that has demonstrated good therapeutic effects in hematologic cancers. METHODS: We used TCGA and GTEx pan-cancer database to evaluate XPO1 mRNA expression in various tumors. Cell proliferation assay and colony formation assay were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. Western blot was performed to explore the specific mechanisms. RESULTS: We found that XPO1 was highly expressed across a range of cancers and associated with poor prognosis in hepatobiliary and pancreatic tumors. We revealed that the XPO1 inhibitor KPT-330 triggered the nuclear accumulation of the p53 protein and significantly disrupted the proliferation of cholangiocarcinoma cells. Mechanistically, the XPO1 inhibitor, KPT-330, reduced BIRC6 expression by inhibiting the PI3K/AKT pathway to decrease p53 degradation and improve its stability. CONCLUSION: Therefore, XPO1 may be a potential therapeutic target in cholangiocarcinoma, mediated by its effects on KPT-330.

Perioperative Outcomes and Learning Curve of Robot-Assisted McKeown Esophagectomy.

BACKGROUND: This study aimed to evaluate the perioperative outcomes of patients undergoing robot-assisted McKeown esophagectomy (RAME) and the learning curves of surgeons performing RAME at a single center. METHODS: Perioperative outcomes of RAME and video-assisted McKeown esophagectomy (VAME) were compared after eliminating confounding factors by propensity score matching (PSM). The cumulative sum (CUSUM) method was used to evaluate the learning curves of RAME for a single surgical team. RESULTS: In general, a total of 198 patients with esophageal cancer (RAME: 45 patients, VAME: 153 patients) were included in this study, and 43 pairs of patients receiving RAME or VAME were matched using 1:1 PSM analysis. Those in the RAME group had more lymph nodes dissected in the total lymph nodes (median 29.0 vs. 26.0, P = 0.011) and the upper mediastinum (median 8.0 vs. 6.0, P < 0.001), especially the left recurrent laryngeal nerve (RLN) lymph node (median 4.0 vs. 2.0, P = 0.001). According to the trend of the CUSUM plot, the learning curve was divided into two stages at the 20th RAME procedure. After mastering the learning curve, RAME harvested a significantly higher number of upper mediastinal lymph nodes (median 9.0 vs. 6.0, P = 0.001), left RLN lymph nodes (median 5.0 vs. 3.5, P = 0.003), and right RLN lymph nodes (median 4.0 vs. 2.0, P = 0.002). Meanwhile, the incidence of postoperative pneumonia in the proficiency phase was significantly lower than that in the learning phase (4.0% vs. 25.0%, P = 0.04). CONCLUSIONS: RAME improved left RLN lymph node dissection. Surgeons with extensive VAME experience need at least 20 cases to achieve early proficiency in RAME.

Inhibition of XPO1 with KPT-330 induces autophagy-dependent apoptosis in gallbladder cancer by activating the p53/mTOR pathway.

BACKGROUND: Gallbladder cancer (GBC) is a highly aggressive malignant cancer in the biliary system with poor prognosis. XPO1 (chromosome region maintenance 1 or CRM1) mediates the nuclear export of several proteins, mainly tumor suppressors. Thus, XPO1 functions as a pro-oncogenic factor. KPT-330 (Selinexor) is a United States Food and Drug Administration approved selective inhibitor of XPO1 that demonstrates good therapeutic effects in hematologic cancers. However, the function of XPO1 and the effect of KPT-330 have not been reported in GBC. METHODS: We analyzed the correlation between XPO1 expression levels by q-PCR and clinical features of GBC patients. Cell proliferation assays were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. mRNA sequencing was used to explore the underlying mechanisms. Western blot was performed to explore the relationship between apoptosis and autophagy. The in vivo antitumor effect of KPT-330 was investigated in a nude mouse model of gallbladder cancer. RESULTS: We found that high expression of XPO1 was related to poor prognosis of GBC patients. We observed that XPO1 inhibitor KPT-330 inhibited the proliferation of GBC cells in vitro. Furthermore, XPO1 inhibitor KPT-330 induced apoptosis by reducing the mitochondrial membrane potential and triggering autophagy in NOZ and GBC-SD cells. Indeed, XPO1 inhibitor KPT-330 led to nuclear accumulation of p53 and activated the p53/mTOR pathway to regulate autophagy-dependent apoptosis. Importantly, KPT-330 suppressed tumor growth with no obvious toxic effects in vivo. CONCLUSION: XPO1 may be a promising prognostic indicator for GBC, and KPT-330 appears to be a potential drug for treating GBC effectively and safely.

UBAP2L promotes gastric cancer metastasis by activating NF-κB through PI3K/AKT pathway.

Ubiquitin-associated protein 2-like (UBAP2L) is highly expressed in various types of tumors and has been shown to participate in tumor growth and metastasis; however, its role in gastric cancer (GC) remains unknown. In this study, we observed that UBAP2L expression was markedly elevated in GC tissues and five GC cell lines. Higher expression of UBAP2L was associated with poor prognosis as revealed by bioinformatics analysis on online websites and laboratory experiments. Knockdown of UBAP2L impeded the migration and invasion abilities of GC cell lines. In contrast, its overexpression enhanced the migration and invasion abilities of GC cell lines. Overexpression of UBAP2L also increased the number and size of lung metastatic nodules in vivo. According to the results of mass spectrometry and pathway annotation of the identified proteins, the PI3K/AKT pathway was found to be related to UBAP2L regulation. Further exploration and rescue experiments revealed that UBAP2L stimulates the expression and nuclear aggregation of p65 and promotes the expression of SP1 by activating the PI3K/AKT pathway. In summary, our findings indicate that UBAP2L regulates GC metastasis through the PI3K/AKT/SP1/NF-κB axis. Thus, targeting UBAP2L may be a potential therapeutic strategy for GC.

Gemcitabine and XCT790, an ERRα inverse agonist, display a synergistic anticancer effect in pancreatic cancer.

Pancreatic cancer (PC) is one of the most fatal and chemoresistant malignancies with a poor prognosis. The current therapeutic options for PC have not achieved satisfactory results due to drug resistance. Therefore, it is urgent to develop novel treatment strategies with enhanced efficacy. This study sought to investigate the anticancer effect of gemcitabine and XCT790, an estrogen-related receptor alpha (ERRα) inverse agonist, as monotherapies or in combination for the treatment of PC. Here we demonstrated that the drug combination synergistically suppressed PC cell viability, its proliferative, migratory, invasive, apoptotic activities, and epithelial-to-mesenchymal transition (EMT), and it triggered G0/G1 cell cycle arrest and programmed cell death in vitro. In addition, in vivo assays using xenograft and mini-PDX (patient-derived xenograft) models further confirmed the synergistic antitumor effect between gemcitabine and XCT790 on PC. Mechanistically, gemcitabine and XCT790 suppressed PC by inhibiting ERRα and MEK/ERK signaling pathway. In conclusion, our current study demonstrated for the first time that gemcitabine combined with XCT790 displayed synergistic anticancer activities against PC, suggesting that their combination might be a promising treatment strategy for the therapy of PC.

Preoperative lymphocyte to C-reactive protein ratio as a new prognostic indicator in patients with resectable gallbladder cancer.

BACKGROUND: Inflammation is often related to cancer, and several inflammatory scores have been established to predict the prognosis of various types of cancer. Our study aimed to determine the prognostic value of the preoperative lymphocyte to C-reactive protein ratio (LCR) for predicting postoperative outcomes in patients with resectable gallbladder cancer (GBC). METHODS: A retrospective analysis of 104 GBC patients who received curative surgery at Xinhua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine from January 2000 to December 2016 was performed. A time-dependent receiver operating characteristic curve was constructed to evaluate the accuracy of different markers. Univariate and multivariate Cox proportional hazard models were used to define factors associated with overall survival. RESULTS: Among the assessed variables, the preoperative LCR showed the highest accuracy in predicting the overall survival of GBC patients (AUC: 0.736). Decreased preoperative LCR was significantly associated with advanced tumor stage, including tumor invasion (P = 0.018), lymph node metastasis (P = 0.011) and TNM stage (P = 0.022). A low preoperative LCR (cutoff threshold = 145.5) was an independent risk factor for overall survival in patients with resectable GBC (P < 0.001). CONCLUSIONS: The preoperative LCR is a novel and valuable prognostic indicator of postoperative survival in patients with resectable GBC.

ESRRA promotes gastric cancer development by regulating the CDC25C/CDK1/CyclinB1 pathway via DSN1.

Background: Estrogen-related receptor-α (ESRRA) is an orphan nuclear receptor, expressing at high level in exuberant metabolism organs and acting as transcription factor. High expression was found in many malignances but no research was done in gastric cancer (GC), where lipid metabolism disorder is common. Methods: Kaplan-Meier plot was utilized to find the relationship between ESRRA expression and patients' prognoses. The expression level of ESRRA was measured by real-time PCR. The protein expression levels were tested with western-blot and immunohistochemistry. Cell cycle and apoptosis was identified with flow cytometry. RNA-seq, bioinformatics analysis, dual-luciferase assay and ChIP assay were used to predict and validate ESRRA's target gene and binding motif. Animal models were also introduced in our study. Results: ESRRA expression is notably higher in GC cell lines and high ESRRA levels are correlated to poor prognoses. ESRRA silencing decreased GC cell viability, migration, and invasion capacities. Its downstream gene DSN1 was spotted by RNA-seq and confirmed by later bioinformatics analyses, dual-luciferase, and ChIP assays. Western-blot showed G2M arrest caused by ESRRA silencing was via CDC25C-CDK1-Cyclin B1 pathway. Conclusion: ESRRA/DSN1/CDC25C-CDK1-Cyclin B1 is of great importance in GC development. ESRRA could be a potential target as well as prognostic marker in GC.

Sensitive and selective detections of mustard gas and its analogues by 4-mercaptocoumarins as fluorescent chemosensors in both solutions and gas phase.

Mustard gas has been used as a chemical warfare agent for a century, and is the most likely chemical weapon used in wars or by terrorists. Thus, it is important to develop a facile, rapid and highly selective method for the detection of mustard gas. In this paper, two fluorescent probe molecules, 4-mercaptocoumarins, have been developed for rapid and sensitive detections of SM and its analogues (CEES and NH1) in both solutions and gas phase. The sensing reaction is a nucleophilic addition at three-membered hetercyclic sulfonium/ammonium formed from SM, CEES/NH1 in ethanol. Two fluorescent probes (4-mercaptocoumarins, ArSH) in ethanol deprotonate to form thiophenol anions (ArS-) resulting from their low pKa values (3.2-3.4), and the nucleophilic addition of the anion ArS- generates the corresponding thioethers, giving a turn-on fluorescence response. The thiophenol anion can fast sense SM, CEES and NH1 (within 1-4 min) with high sensitivity (~nM level) at 60 °C, and high selectivity through adding a tertiary amine, and two probes exhibit excellent chemical and photostability in detection systems. Furthermore, a facile test strip with the sensor was fabricated for the detection of CEES vapor with rapid response (3 min), high sensitivity (9 ppb) and high selectivity.

Quinoline-2-thione-based fluorescent probes for selective and sensitive detections of mustard gas and its analogues.

Sulfur mustard (SM, also called as mustard gas (HD)) is a persistent and highly toxic gas used as chemical weapon in wars and military conflicts. Moreover, owing to its simple structure and easy synthesis, it is the most likely chemical agent used by terrorists. For this reason, it is vital important to develop a facile, rapid and reliable detection system for SM. In this paper, we have developed four quinoline-2-thiones as fluorescent probes, 2a-2d, for the detection of SM and its analogues, half sulfur mustard (CEES) and a nitrogen mustard NH1. In the presence of KOH, these quinoline-2-thiones deprotonated to quinoline-2-thiophenol anions, which react with SM and its analogues rapidly to form quinoline-2-thiethers with highly efficient fluorescence, giving turn-on fluorescence response. The sensing products with CEES were isolated and fully characterized, thereby, the sensing mechanism was firmly established. The fluorescent probes with 4-trifluoromethyl group, 2b and 2d, exhibit rapid response to SM, CEES and NH1 (within 1 min at 60 °C for CEES and NH1), high sensitivity (limit of detection, 50 nM for SM and 20 nM for NH1) and high selectivity. Furthermore, polymer film test strips were fabricated with probe-embedded poly(ethylene oxide) for the detection of CEES vapor. These test strips displayed a rapid response (<4 min) to gaseous CEES with high sensitivity (0.2 ppm) and high selectivity. These results show that fluorescent probes 2b and 2d have a good application prospect in the field detection of mustard gas.

DGCR5 is activated by PAX5 and promotes pancreatic cancer via targeting miR-3163/TOP2A and activating Wnt/ß-catenin pathway.

Long noncoding RNA DiGeorge syndrome critical region gene 5 (DGCR5) has been shown to be highly associated with cancer development. However, the biological role and molecular mechanism of DGCR5 in pancreatic cancer (PC) remains largely unknown. This study aimed to explore the role of DGCR5 in PC. It was revealed that DGCR5 was highly expressed in PC tissues compared with adjacent normal tissues and was associated with poor prognosis in PC patients. Furthermore, DGCR5 depletion inhibited the proliferation, migration and invasion by increasing apoptosis and inducing G0/G1 cell cycle arrest in vitro. Moreover, xenograft assay validated that DGCR5 promotes PC tumor growth in vivo. Mechanistically, DGCR5 was found to act as a ceRNA by sponging miR-3163 to regulate DNA topoisomerase 2-alpha (TOP2A) and inhibit Wnt/ß-catenin pathway. In addition, it was found that DGCR5 downregulation could enhance the sensitivity of PC cells to gemcitabine, and ChIP assay showed that PAX5 (Paired Box 5) could bind to the promoter region of DGCR5 and increase its transcription. The results of the present study indicated that DGCR5 may be a potential diagnostic biomarker and therapeutic target for PC.

A phase III study on neoadjuvant chemotherapy versus neoadjuvant toripalimab plus chemotherapy for locally advanced esophageal squamous cell carcinoma: Henan Cancer Hospital Thoracic Oncology Group 1909 (HCHTOG1909).

BACKGROUND: Neoadjuvant chemotherapy (NAC) and neoadjuvant chemoradiotherapy (NACR) are the standard treatments for esophageal squamous cell carcinoma (ESCC). However, the 5-year overall survival (OS) rate is still far from satisfactory. In recent years, immune checkpoint inhibitors (ICIs) have shown promising results in the treatment of ESCC. More than 20 phase II clinical trials have been launched to explore combinations of ICIs in the neoadjuvant setting for ESCC. Based on our phase II clinical trial, a two-arm phase III trial was launched in Henan Cancer Hospital. ICIs combined with NAC may usher in a new era and may benefit locally advanced, resectable ESCC patients. METHODS: A two-arm phase III trial was launched in April 2020 in Henan Cancer Hospital. Patient recruitment will be completed within 18 months. The primary endpoint is event-free survival (EFS). The secondary endpoints include pathologic complete response (pCR), disease-free survival (DFS) rate, overall response rate (ORR), R0 resection rate, major pathologic response (MPR), adverse events (AEs), complication rate and quality of life (QOL). A biobank of pretreatment, resected tumor tissue and paired blood samples will be built for translational research in the future. DISCUSSION: This RCT directly compares NAC with neoadjuvant toripalimab plus chemotherapy in terms of EFS for locally advanced ESCC. The results may usher in a new era of resectable ESCC treatment. TRIAL REGISTRATION: NCT04280822 (https://www.clinicaltrials.gov/ct2/show/NCT04280822). Registered title: "A Phase III, Randomized Controlled Study of Neo-adjuvant Toripalimab (JS001) in Combination with Chemotherapy versus Neo-adjuvant Chemotherapy for Resectable Esophageal Squamous Cell Carcinoma". Version 1.0/Nov. 21, 2019.

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS. The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion. Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1 ng/mL in PBS and 0.5 ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300 ng/mL in PBS, 1.0-400 ng/mL in milk, and 1.0-250 ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.