Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/ß splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising candidate for treating metabolic disorder diseases and has been used in phase II clinical trials. Currently, metabolic diseases are prevalent worldwide, underscoring the significant market potential of FGF21. Therefore, the production of FGF21 must be effectively improved to meet market demand. RESULTS: Herein, to investigate the impact of vectors and host cells on FGF21 expression, we successfully engineered strains that exhibit a high yield of FGF21. Surprisingly, the data revealed that vectors with various copy numbers significantly impact the expression of FGF21, and the results showed a 4.35-fold increase in expression levels. Furthermore, the performance of the double promoter and tandem gene expression construction design surpassed that of the conventional construction method, with a maximum difference of 2.67 times. CONCLUSION: By exploring engineered vectors and host cells, we successfully achieved high-yield production of the FGF21 strain. This breakthrough lays a solid foundation for the future industrialization of FGF21. Additionally, FGF21 can be easily, quickly and efficiently expressed, providing a better tool and platform for the research and application of more recombinant proteins.

Development of an Integrated Platform for the Simultaneous Enrichment and Characterization of N- and O-Linked Intact Glycopeptides.

Both N-linked glycosylation and O-linked glycosylation play essential roles in the onset and progression of various diseases including cancer, and N-/O-linked site-specific glycans have been proven to be promising biomarkers for the discrimination of cancer. However, the micro-heterogeneity and low abundance nature of N-/O-linked glycosylation, as well as the time-consuming and tedious procedures for the enrichment of O-linked intact glycopeptides, pose great challenges for their efficient and accurate characterization. In this study, we developed an integrated platform for the simultaneous enrichment and characterization of N- and O-linked intact glycopeptides from the same serum sample. By fine-tuning the experimental conditions, we demonstrated that this platform allowed the selective separation of N- and O-linked intact glycopeptides into two fractions, with 85.1% O-linked intact glycopeptides presented in the first fraction and 93.4% N-linked intact glycopeptides presented in the second fraction. Determined with high reproducibility, this platform was further applied to the differential analysis of serum samples of gastric cancer and health control, which revealed 17 and 181 significantly changed O-linked and N-linked intact glycopeptides. Interestingly, five glycoproteins containing both significant regulation of N- and O-glycosylation were observed, hinting potential co-regulation of different types of glycosylation during tumor progress. In summary, this integrated platform opened a potentially useful avenue for the global analysis of protein glycosylation and can serve as a useful tool for the characterization of N-/O-linked intact glycopeptides at the proteomics scale.

Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional liquid chromatography-mass spectrometry based on a novel phthalic anhydride bonded stationary phase.

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional liquid chromatography-mass spectrometry was developed. In this two-dimensional liquid chromatography system, a novel phthalic anhydride-bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional liquid chromatography-mass spectrometry, this online two-dimensional liquid chromatography-mass spectrometry system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines.

Combining multidimensional chromatography-mass spectrometry and feature-based molecular networking methods for the systematic characterization of compounds in the supercritical fluid extract of Tripterygium wilfordii Hook F.

Tripterygium wilfordii Hook F from the family Celastraceae is a traditional Chinese medicine (TCM) whose principal chemical constituents are terpenoids, including sesquiterpene alkaloids and diterpenoids, which have unique and diverse structures and remarkable biological activities. In order to advance pharmacological research and guide the preparation of monomer compounds derived from T. wilfordii, a systematic approach to efficiently discover new compounds or their derivatives is needed. Herein, compound separation and identification were performed by offline reversed-phase × supercritical fluid chromatography coupled mass spectrometry (RP × SFC-Q-TOF-MS/MS) and Global Natural Product Social (GNPS) molecular networking. The 2D chromatography system exhibited a high degree of orthogonality and significant peak capacity, and SFC has an advantage during the separation of sesquiterpene alkaloid isomers. Feature-based molecular networking offers the great advantage of quickly detecting and clustering unknown compounds, which greatly assists in intuitively judging the type of compound, and this networking technique has the potential to dramatically accelerate the identification and characterization of compounds from natural sources. A total of 324 compounds were identified and quantitated, including 284 alkaloids, 22 diterpenoids and 18 triterpenoids, which means that there are numerous potential new compounds with novel structures to be further explored. Overall, feature-based molecular networking provides an effective method for discovering and characterizing novel compounds and guides the separation and preparation of targeted natural products.

High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase.

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15 mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R2  > 0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87-110.99%), limits of detection (5.35-5.76 µg/mL), and limits of quantification (20 µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents of three batches of Ephedrae Herba from three pharmaceutical companies.

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.

Preparative chromatography behavior of palmitoleic acid on octadecyl stationary phases with different densities.

The availability of various high-purity unsaturated fatty acids has a wide range of needs due to their different activities. The nonlinear preparative chromatography behavior and principle for purification of palmitoleic acid with octadecyl bonded stationary phases were studied. The peak broadening and the concentration distribution of the target compounds were used to compare different C18 stationary phases. In preparative liquid chromatography, the C18 stationary phases with low, medium, and high bonding density showed different peak broadening and concentration distribution results. Medium bonding density C18 was suitable for the purification of ethyl palmitoleic acid. The forward broadening was much greater than the backward broadening on medium bonding density C18 column. And the highest concentration distribution of impurities and the main peak was not crossed in this column. Due to the low content of crude ethyl palmitoleic acid sample, a two-step purified method yields an oily product with purity of 96.57% in the GC method. This method would be universal and extensible for constructing purification method for other unsaturated fatty acids.

Comprehensive profiling of Sanguisorba officinalis using offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography, tandem high-resolution mass spectrometry, and molecular network.

The profiling of natural products is important in modern biological sciences and new drug development. However, the separation and characterization of complex herbal extracts are significantly challenging for researchers in the biochemical field. Herein, an offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography system is developed. Our system exhibits high orthogonality and is composed of a newly prepared stationary phase in the first dimension and a traditional C18 phase in the second dimension, and is operated in combination with a high-resolution mass spectrometry and molecular network. Sanguisorba officinalis L. is studied using the proposed method owing to its bioactivity. With the aid of orthogonal separation, the ionization of the individual components is improved. The number of detected compounds and separated peaks are significantly increased when one-dimensional liquid chromatography is upgraded to two-dimensional liquid chromatography. In addition, 270 compounds (127 of which are tentatively characterized as new compounds, and further confirmation is needed) are successfully characterized based on their fragmentation patterns under the guidance of molecular network, while only 95 compounds are characterized using one-dimensional liquid chromatography and high-resolution mass spectrometry. The results indicate that the developed offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography, tandem high-resolution mass spectrometry, and molecular network method are effective for profiling complex samples.

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure-Activity Relationship and Inhibition Profiles.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration-response curves. A remarkable structure-activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non-aggregated state in solution and a high inhibition potency (IC50 = 0.50 ± 0.08 µM), and therefore was selected as a representative for the ligand-protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high-potency ACLY inhibitors. In-depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.

An Integrated Study on the Fading Mechanism of Malachite Green Industrial Dye for the Marquisette Curtain in the Studio of Cleansing Fragrance, the Palace Museum (Beijing).

Historical marquisette curtains were composed of lightweight fabrics, woven in an open-mesh and leno-type weave, usually made of silk, and found in Qing imperial buildings. As panel curtains, they were exposed to light, and so underwent fading. This study investigated the manufacturing technology and fading mechanism of dyed marquisette fabric from the Studio of Cleansing Fragrance, the Palace Museum (Beijing). The technological aspects were identified. The types of weave, fiber, and adhesive used to fix the curtain to the wooden frame were identified through microscopic observation and infrared spectroscopy. A color change characterization was performed based on UV-visible diffuse reflectance spectra. The textile colorant was identified as malachite green (MG), and its degradation by light was subsequently studied by dynamic photolysis experiments in a kinetic solution for the rapid exploration of by-products. The main degradation pathways were thus identified and the factors responsible for the induced color changes were discussed. A comparison of the liquid chromatography-mass spectrometry (LC-MS) results of the products derived from the photolysis method as well as of the samples extracted from the object allowed for the identification of the presence of different degradation pathways in the faded and unfaded parts of the textile. A metabolomics analysis was applied to account for the differences in the degradation pathways.

Integration of Two-Dimensional Liquid Chromatography-Mass Spectrometry and Molecular Docking to Characterize and Predict Polar Active Compounds in Curcuma kwangsiensis.

Curcuma kwangsiensis, one species of Curcumae zedoaria Ros. c, is a commonly used traditional Chinese medicine (TCM) for treating cardiovascular disease, cancer, asthma and inflammation. Polar compounds are abundant in water decoction, which would be responsible for critical pharmacological effects. However, current research on polar compounds in Curcumae zedoaria Ros. c remains scarce. In this study, the polar fraction from Curcuma kwangsiensis was firstly profiled on G protein-coupled receptor 109A (GPR109A), ß2-adrenergic receptor (ß2-AR), neurotensin receptor (NTSR), muscarinic-3 acetylcholine receptor (M3) and G protein-coupled receptor 35 (GPR35), which were involved in its clinical indications and exhibited excellent ß2-AR and GPR109A receptor activities. Then, an offline two-dimensional reversed-phase liquid chromatography (RPLC) coupled with the hydrophilic interaction chromatography (HILIC) method was developed to separate polar compounds. By the combination of a polar-copolymerized XAqua C18 column and an amide-bonded XAmide column, an orthogonality of 47.6% was achieved. As a result of coupling with the mass spectrometry (MS), a four-dimensional data plot was presented in which 373 mass peaks were detected and 22 polar compounds tentatively identified, including the GPR109A agonist niacin. Finally, molecular docking of these 22 identified compounds to ß2-AR, M3, GPR35 and GPR109A receptors was performed to predict potential active ingredients, and compound 9 was predicted to have a similar interaction to the ß2-AR partial agonist salmeterol. These results were supplementary to the material basis of Curcuma kwangsiensis and facilitated the bioactivity research of polar compounds. The integration of RPLC×HILIC-MS and molecular docking can be a powerful tool for characterizing and predicting polar active components in TCM.

Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome.

Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.

A strategy for efficient enrichment of steroidal alkaloids from Fritillaria based on fluorinated reversed-phase stationary phase.

Plant-derived alkaloids are bioactive natural ingredients, but their contents are relatively low in plants. Therefore, the efficient enrichment of alkaloids is a prerequisite for purification and further pharmacological research. In this study, an efficient and simple strategy for enrichment of steroidal alkaloids in Fritillaria was developed for the first time based on the fluorinated reverse-phase stationary phase (FC8HL). Superior selectivity between alkaloids and non-alkaloids was achieved in a non-aqueous system, and a simple solvent system containing low-content additives was applied to elute alkaloids. Key parameters that affected the elution were investigated, including different types of buffer salts and optimized concentrations. The optimized elution system was then applied to selectively enrich alkaloids from five species of Fritillaria. Its practicability was further demonstrated by enrichment of alkaloids from Fritillaria cirrhosa D.Don at a preparative level. This developed method has great potential for other types of hydrophobic alkaloids.

Application of two-dimensional liquid chromatography in the separation of traditional Chinese medicine.

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two-dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one-dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two-dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two-dimensional liquid chromatography, heart-cutting two-dimensional liquid chromatography both in on-line, and off-line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two-dimensional liquid chromatography.

Determination of glycosylation degree for glycoconjugate vaccines using a solid-phase extraction combined with liquid chromatography and tandem mass spectrometry method.

In this study, a solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed to determine the degree of glycosylation of glycosylation sites and the ratio of free carrier protein to total carrier protein for glycoconjugate vaccines. To remove and enrich the glycosylated peptides, a solid-phase extraction method was developed, optimized, and hyphenated to liquid chromatography-tandem mass spectrometry. The developed solid-phase extraction with liquid chromatography-tandem mass spectrometry method was shown to possess a wide linear dynamic range (0.03-100 µg/mL), a high sensitivity (0.03 µg/mL for CRM197), good interday and intra-day precision (relative standard deviation of peak area < 3.3%), and good recoveries from vaccine matrix (90-105%). Finally, the method was utilized to determine the degree of glycosylation and free carrier protein to total carrier protein ratio for pneumococcal conjugate vaccines and meningococcal vaccines. For quality evaluation of glycoconjugate vaccines, the method could provide more information than the traditional size exclusion chromatography method. Fourteen and twelve reported glycosylation sites for CRM197- and tetanus toxin-based vaccines can be detected, respectively.

Highly Efficient Analysis of Glycoprotein Sialylation in Human Serum by Simultaneous Quantification of Glycosites and Site-Specific Glycoforms.

Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of these diseases. However, in-depth analysis of sialylation is still challenging because of the high microheterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detailed characterization of glycoprotein sialylation on the levels of glycosites and site-specific glycoforms by employing the SGP enrichment method. This strategy enabled the identification of up to 380 glycosites, as well as 414 intact glycopeptides corresponding to 383 site-specific glycoforms from only initial 6 µL serum samples, indicating the high sensitivity of the method for the detailed analysis of glycoprotein sialylation. This strategy was further employed to the differential analysis of glycoprotein sialylation between hepatocellular carcinoma patients and control samples, leading to the quantification of 344 glycosites and 405 site-specific glycoforms, simultaneously. Among these, 43 glycosites and 55 site-specific glycoforms were found to have significant change on the glycosite and site-specific glycoform levels, respectively. Interestingly, several glycoforms attached onto the same glycosite were found with different change tendencies. This strategy was demonstrated to be a powerful tool to reveal subtle differences of the macro- and microheterogeneity of glycoprotein sialylation.

Chemoenzymatic Synthesis of O-Mannose Glycans Containing Sulfated or Nonsulfated HNK-1 Epitope.

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on O- and N-glycans of various glycoproteins and on glycolipids. It is overexpressed in the nervous system and plays crucial roles in nerve regeneration, synaptic plasticity, and neuronal diseases. However, the investigation of functional roles of HNK-1 in a more complex glycan context at the molecular level remains a big challenge due to lack of access to related structurally well-defined complex glycans. Herein, we describe a highly efficient chemoenzymatic approach for the first collective synthesis of HNK-1-bearing O-mannose glycans with different branching patterns, and for their nonsulfated counterparts. The successful strategy relies on both chemical glycosylation of a trisaccharide lactone donor for the introduction of sulfated HNK-1 branch and substrate promiscuities of bacterial glycosyltransferases that can tolerate sulfated substrates for enzymatic diversification. Glycan microarray analysis with the resulting complex synthetic glycans demonstrated their recognition by two HNK-1-specific antibodies including anti-HNK-1/N-CAM (CD57) and Cat-315, which provided further evidence for the recognition epitopes of these antibodies and the essential roles of the sulfate group for HNK-1 glycan-antibody recognition.

Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry.

Human milk oligosaccharides (HMOs) are one of the most abundant ingredients in breast milk, and they play a beneficial role for newborns and are important for infant health. The peripheral fucosylated sequences of HMOs, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens, are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland, and are often the recognition motifs and serve as decoy receptors for microbes. In this work, we developed a method for determination of secretor status and Lewis blood phenotype and assignment of Lewis blood-group epitopes. The method was based on electrostatic repulsion/hydrophilic interaction chromatography coupled with tandem mass spectrometry (ERLIC-MS/MS). A specifically designed stationary phase, aspartic acid-bonded silica (ABS), was used to separate the acidic and neutral HMOs by electrostatic repulsion followed by HILIC. Negative-ion electrospray MS/MS was then used for analysis of secretor status and Lewis blood phenotypes and assignment of important epitopes of HMOs from the lactating mothers by selecting a specific set of unique fragment ions.

Two Compounds Isolated From Ganglioside GM1 Promote Angiogenesis in Zebrafish.

Ganglioside has been implicated to play important roles in modulating various cell signaling and biological functions. However, the functional analysis of a single ganglioside in a zebrafish model is so far lacking. In this study, we investigated the angiogenic effects of 2 monosialoganglioside compounds isolated from GM1 in zebrafish embryos. First, we showed the tested compounds are adequate safe. Then, we found that these compounds exhibited significant proangiogenic effect through enhancement of endothelial cell proliferation, migration, and differentiation. Furthermore, the 2 compounds were proved to promote angiogenesis through, at least partially, modulating the level of Notch signaling. This study provides the novel insights into the clinical application of the 2 ganglioside compounds and GM1.