Fast Discrimination of Sialylated N-Glycan Linkage Isomers with One-Step Derivatization by Microfluidic Capillary Electrophoresis-Mass Spectrometry.

Linkage isomers (α-2,3- or α-2,6-linkage) of sialylated N-glycans are involved in the emergence and progression of some diseases, so they are of great significance for diagnosing and monitoring diseases. However, the qualitative and quantitative analysis of sialylated N-glycan linkage isomers remains challenging due to their low abundance and limited isomeric separation techniques. Herein, we developed a novel strategy integrating one-step sialic acid derivatization, positive charge-sensitive separation and highly sensitive detection based on microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for fast and specific analysis of α-2,3- and α-2,6-linked sialylated N-glycan isomers. A kind of easily charged long-chain amino compound was screened first for one-step sialic acid derivatization so that only α-2,3- and α-2,6-linked isomers can be quickly and efficiently separated within 10 min by MCE due to the difference in structural conformation, whose separation mechanism was further theoretically supported by molecular dynamic simulation. In addition, different sialylated N-glycans were separated in order according to the number of sialic acids, so that a migration time-based prediction of the number of sialic acids was achieved. Finally, the sialylated N-glycome of human serum was profiled within 10 min and 6 of the 52 detected sialylated N-glycans could be potential diagnostic biomarkers of cervical cancer (CC), whose α-2,3- and α-2,6-linked isomers were distinguished by α-2,3Neuraminidase S.

Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis-Mass Spectrometry.

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking.

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross-linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short-end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis-Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half-maximal inhibitory concentration of kojic acid was 5.55 µM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study.

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis-Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.

Rapid proteolytic digestion and peptide separation using monolithic enzyme microreactor coupled with capillary electrophoresis.

Proteolytic digestion and peptide separation are key steps in proteomic study, which has attracted much attention. A novel immobilized trypsin microreactor based on monolithic capillary column was developed for rapid proteolytic digestion in this work. The monolithic support was prepared using itaconic acid as functional monomer within a silanized fused-silica capillary. By taking advantage of itaconic acid with two carboxylic functional groups, trypsin was covalently immobilized onto the monolithic support by condensation reaction. The performance of proteolytic digestion by the microreactor was evaluated with cytochrome C and bovine serum albumin, and the digests were further analyzed by CE and HPLC. Compared to those obtained by conventional in-solution digestion, the digestion time was shortened from 12 h to 40 s, demonstrating the high digestion efficiency of the prepared microreactor. Difference of protein separation results obtained by CE and HPLC highlighted the potential of CE in fast and highly efficient peptide separation in proteomic study. All the results demonstrated that the microreactor combined with CE could be a promising tool in workflow of proteomic analysis.

Association of exposure to multiple metals with papillary thyroid cancer risk in China.

Papillary thyroid cancer (PTC) has inflicted huge threats to the health of mankind. Metal pollution could be a potential risk factor of PTC occurrence, but existing relevant epidemiological researches are limited. The current case-control study was designed to evaluate the relationships between exposure to multiple metals and the risk of PTC. A total of 262 histologically confirmed PTC cases were recruited. Age- and gender-matched controls were enrolled at the same time. Urine samples were used as biomarkers to reflect the levels of environmental exposure to 13 metals. Conditional logistic regression models were adopted to assess the potential association. Single-metal and multi-metal models were separately conducted to evaluate the impacts of single and co-exposure to 13 metals. The increased concentration of urinary Cd, Cu, Fe, and Pb quartiles was found significant correlated with PTC risk. We also found the decreased trends of urinary Se, Zn, and Mn quartiles with the ORs for PTC. These dose-response associations between Pb and PTC were observed in the single-metal model and remained significant in the multi-metal model (OR25-50th=1.39, OR50-75th=3.32, OR>75th=7.62, p for trend <0.001). Our study suggested that PTC was positively associated with urinary levels of Cd, Cu, Fe, Pb, and inversely associated with Se, Zn, and Mn. Targeted public health policies should be made to improve the environment and the recognition of potential risk factors. These findings need additional studies to confirm in other population.

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis.

Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis (EMMA) and immobilized enzyme microreactor (IMER) are summarized in this article.

Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory.

Two purified polysaccharides named WPMP-1 and WPMP-2 were obtained from Polygonum multiflorum with an average molecular weight of 2.04kDa and 92.13kDa, respectively. Structural characterization revealed that WPMP-1was supposed to be a glucan composed of 1,4-linked α-d-Glcp, 1,4,6-linked α-d-Glcp, and terminal α-d-Glcp. WPMP-2 contained a complex branched structre formed by 1,2-linked α-d-Rhap, 1,2,4-linked α-d-Rhap, 1,3,5-linked α-l-Ara, 1,5- linked α-l-Araf, terminal α-l-Araf, 1,4-linked ß-d-Galp, 1,4-linked α-d-GalpA, and 4-linked α-d-GalpA. In vitro, WPMPs were of activation effect on splenocyte and macrophages, and could also protect immunocyte against 5-Fu induced immunosupression by restoring the proliferation rate, phagocytic index and cytokines secretion level. Moreover, the acid polysaccharide WPMP-2 exhibited better immunomodulatory activity than neutral polysaccharide WPMP-1, which indicated that the immunomodulatory activity of WPMPs were positively associated with higher content of uronic acid, percentage of rhamnose, arabinose and galactose, and branching degree. These findings could promote the polysaccharides from Polygonum multiflorum to be attractive functional food supplement and immunomodulatory adjuvant.

New utilization of Polygonum multiflorum polysaccharide as macromolecular carrier of 5-fluorouracil for controlled release and immunoprotection.

WPMP-2 is an acid polysaccharide isolated from Polygonum multiflorum, which demonstrated excellent immunomodulatory activity. In order to reduce immunosuppression of 5-fluorouracil (5-Fu), WPMP-2 was utilized as a macromolecular carrier to conjugate the 5-Fu derivatives 5-fluorouracil-1-acetic acid (5-FUAC) through ester bond. The conjugate showed controlled drug release behaviour in vitro at 37°C in phosphate buffer (pH7.4), and only 5-FUAC was detected in the media. The cytotoxicity test in vitro showed that the conjugate exhibited different cytotoxicity to HepG-2 and HT-29 cells. In addition, immunization study in vivo illustrated that the conjugate displayed immunoprotective effect by mitigating inhibition and damage effects of 5-Fu on secretion of cytokines, proliferation of splenocytes, and phagocytosis of peritoneal macrophages. It was indicated that the conjugation of 5-Fu and WPMP-2 could be a potential double effective drug delivery system.