Recent bioanalytical methods for the isolation of bioactive natural products from genus Codonopsis.

INTRODUCTION: Chromatography and spectroscopy are nowadays well-validated techniques allowing to isolate and purify different class of natural products from the genus Codonopsis. Several categories of phytochemicals with drug like properties have been selectively extracted, isolated, characterised by this methodology. OBJECTIVES: The present review aims to provide up-to-date and comprehensive information on the chromatography, phytochemistry and pharmacology of natural products of Codonopsis with an emphasis on the search for natural products having various biological activities and the semi-synthetic derivatives of bioactive ones and to highlight current gaps in knowledge. MATERIALS AND METHODS: A literature search was performed in the SciFinder Scholar, PubMed, Medline, and Scopus databases. RESULTS: During the period covered in this review, several classes of compounds have been reported from genus Codonopsis. Codonopsis pilosula and Codonopsis lanceolata are the most popular in the genus especially as per phytochemical and bioactive studies. Phytochemical investigation demonstrates that Codonopsis species contain mainly xanthones, flavonoids, alkaloids, polyacetylenes, phenylpropanoids, triterpenoids and polysaccharides, which contribute to numerous bioactivities. The major bioactive compounds isolated were used for semi-synthetic modification to increase the chance to discover lead compound. CONCLUSIONS: It can be concluded that genus Codonopsis has been used as traditional medicines and food materials around the world over years due to chemical constituents with diverse structural types, exhibiting extensive pharmacological activities in immune system, blood system, cardiovascular system, central nervous system, digestive system, and so forth, with almost no obvious toxicity and side effect. Therefore, Codonopsis can be used as a promising ethnopharmacological plant source.

An integrated proteomic and glycoproteomic study for differences on glycosylation occupancy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease in which certain immune cells are dysfunctional and attack their own healthy tissues. There has been great difficulty in finding an accurate and efficient method for the diagnosis of early-stage RA. The present shortage of diagnostic methods leads to the rough treatments of the patients in the late stages, such as joint removing. Nowadays, there is an increasing focus on glyco-biomarkers discovery for malicious disease via MS-based strategy. In this study, we present an integrated proteomics and glycoproteomics approach to uncover the pathological changes of some RA-related glyco-biomarkers and glyco-checkpoints involved in the RA onset. Among 39 distinctly expressive N-glycoproteins, 27 N-glycoproteins were discovered with over twofold expression significances. On the other hand, 13 proteins have been distinguished with significant differences in 53 distinctly expressed proteins identified in this study. Such an integrated approach will provide a comprehensive strategy for new potential glyco-biomarkers and checkpoints discovery in rheumatoid arthritis.

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry.

Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP-LC-MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D'(TIL'-E') showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics.

Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.

N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H218O. Subsequent LC-MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification. Graphical abstract Proposed mechanism of glycopeptides enrichment using DEAE-Sepharose.

Effects of Plant-Growth-Promoting Rhizobacteria (PGPR) and Cyanobacteria on Botanical Characteristics of Tomato (Solanum lycopersicon L.) Plants.

Tomatoes are an important agricultural product because they contain high concentrations of bioactive substances, such as folate, ascorbate, polyphenols, and carotenoids, as well as many other essential elements. As a result, tomatoes are thought to be extremely beneficial to human health. Chemical fertilizers and insecticides are routinely utilized to maximize tomato production. In this context, microbial inoculations, particularly those containing PGPR, may be utilized in place of chemical fertilizers and pesticides. In this study, we investigated the effects of PGPR (Bacillus subtilis, and Bacillus amyloliquefaciens) and cyanobacteria when utilized alone, and in conjunction with each other, on the growth, quality, and yield of fresh fruits of tomato plants. The results showed that the inoculation significantly increased all measured parameters of tomato plants compared with the control. Combined use of B. subtilis and B. amyloliquefaciens had a positive impact on tomato yield, increasing fruit yield. Moreover, leaflet anatomical characteristics were altered, with increased thickness of the upper epidermis, lower epidermis, palisade tissue, spongy tissue, and vascular bundles. Tomato fruit quality was improved, as measured by an increased number of fruit per plant (76% increase), fruit weight (g; 33% increase), fruit height (cm; 50% increase), fruit diameter (cm; 50%), total soluble solids (TSS; 26% increase), and ascorbic acid (mg/100 g F.W.; 75% increase), relative to the control, in the first season. In addition, fruit chemical contents (N, P, and K) were increased with inoculation. The results suggest that inoculation with B. subtilis and B. amyloliquefaciens could be successfully used to enhance tomato plant growth and yield.

Site-Specific N-Glycosylation on the AAV8 Capsid Protein.

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS.

Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the "glycocodes" requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (∼99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitted direct discrimination of α2,3- and α2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both α2,3- and α2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.

Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation.

UNLABELLED: Core-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma. We found that alternating HCD and ETD fragmentation (AHEF) can provide a complementary method to discover CF-glycosites. A total of 407 CF-glycosites among 267 CF-glycoproteins were identified in a mixed sample made from six normal human plasma samples. Among the 407 CF-glycosites, 10 are without the N-X-S/T/C consensus motif, representing 2.5% of the total number identified. All identified CF-glycopeptide results from HCD and ETD fragmentation were filtered with neutral loss peaks and characteristic ions of GlcNAc from HCD spectra, which assured the credibility of the results. This study provides an effective method for CF-glycosites identification and a valuable biomarker reference for clinical research. BIOLOGICAL SIGNIFICANCE: CF-glycosytion plays an important role in regulating biological processes in eukaryotes. Alterations of the glycosites and attached CF-glycans are frequently observed in various types of cancers. Thus, it is crucial to develop a strategy for mapping human CF-glycosylation. Here, we developed a complementary method via alternating HCD and ETD fragmentation (AHEF) to analyze CF-glycoproteins. This strategy reveals an excellent complementarity of HCD and ETD in the analysis of CF-glycoproteins, and provides a valuable biomarker reference for clinical research.