Linjuan Huang

Isolation and structural characterization of the polysaccharide LRGP1 from Lycium ruthenicum.

A novel water-soluble glycoconjugate, designated as Lycium ruthenicum glycoconjugate polysaccharide 1 (LRGP1), was isolated from the fruits of Lycium ruthenicum Murr. The crude polysaccharide was obtained by hot water extraction and purified by ion-exchange and gelfiltration chromatography. Its molecular weight was 56.2 kDa determined by HPGPC (high performance gel permeation chromatography). Monosaccharide composition analysis revealed that it was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.65:10.71:0.33:0.67:1:10.41. The existence of O-type carbohydrate-peptide linkage in LRGP1 was demonstrated by β-elimination reaction. On the basis of monosaccharide composition, partial acid hydrolysis, methylation analysis and ESI-MS analysis, LRGP1 was characterized as a branched polysaccharide rich in arabinose and galactose with a backbone composed of (1→3)-linked Gal. The branches were composed of (1→5)-linked Ara, (1→2)-linked Ara, (1→6)-linked Gal, (1→3)-linked Gal, (1→4)-linked Gal and (1→2,4)-linked Rha. Arabinose, xylose, mannose, and glucose were located at the terminal of the branches.

Structural characterization and antioxidant activities of κ-carrageenan oligosaccharides degraded by different methods.

In the present study, four kinds of κ-carrageenan oligosaccharides were obtained by the degradation of parent κ-carrageenan using free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, respectively. Their structure types were accurately and comparatively elucidated by ESI-MS and CID MS/MS. The antioxidant activities of different degraded products were investigated by four different antioxidant assays, including superoxide radical scavenging activity, hydroxyl radical scavenging activity, reducing power and DPPH radical scavenging activity. The methods of depolymerization had an influence on the antioxidant activities of κ-carrageenan oligosaccharides obtained from κ-carrageenan. These results indicated that the antioxidant activities of κ-carrageenan oligosaccharides could be related to the degree of polymerization, the content of reducing sugar and sulfate groups, and the structure of reducing terminus.

Structural characterization of LbGp1 from the fruits of Lycium barbarum L.

A water-soluble polysaccharide with a protein content of 3.75%, designated as LbGp1, was isolated from the fruit of Lycium barbarum L. The average molecular weight of LbGp1 was 49.1 KDa determined by high performance gel permeation chromatography (HPGPC). Sugar composition analysis revealed that it was composed of Ara and Gal in a molar ratio of 5.6:1. The existence of O-glycopeptide bond in LbGp1 was demonstrated by β-elimination reaction. Structure of LbGp1 was characterized based on monosaccharide composition, partial hydrolysis, methylation analysis and electrospray ionization mass spectrometer (ESI-MS). LbGp1 was identified to be a highly branched polysaccharide with a backbone of →6)Galp(1→linked galactose substituted at O-3 by galactosyl or arabinosyl groups. The branches were composed of (1→3)-linked-Galp, (1→4)-linked-Galp, (1→2)-linked-Araf and (1→3)-linked-Araf, and arabinose was located at the terminal of the branches.

Isolation and structural characterization of a polysaccharide LRP4-A from Lycium ruthenicum Murr.

A complex polysaccharide, termed LRP4-A, was isolated from the fruit of Lycium ruthenicum Murr. and its structure was characterized. The crude polysaccharide LRP was obtained from the fruit of L. ruthenicum Murr. using hot water extraction followed by ethanol precipitation. The water-soluble polysaccharide LRP4-A was purified from LRP by anion-exchange chromatography and gel filtration chromatography. Its molecular weight was 1.05×10(5) Da. Monosaccharide composition analysis revealed that LRP4-A mainly consisted of rhamnose, arabinose, glucose, and galactose in the molar ratio of 1:7.6:0.5:8.6, with a trace of xylose. Structure of the polysaccharide LRP4-A was characterized using a series of analytical techniques, including methylation analysis, partial acid hydrolysis, IR, NMR, and ESI-MS. LRP4-A was identified to be a highly branching polysaccharide with a backbone of β-(1→6)-linked galactose partially substituted at O-3 position. The branches were composed of (1→3)-linked-Gal, (1→3)-linked-Ara, (1→5)-linked-Ara, and (1→2,4)-linked-Rha. Arabinose, galactose, and glucose were located at the termini of the branches.

Characterization of an immunologically active pectin from the fruits of Lycium ruthenicum

An immunologically active pectin, named LRGP5, was firstly isolated from the fruits of Lycium ruthenicum Murr. It contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:2.2:0.5:1.2:4.7. Its molecular weight was estimated to be approximately 1.37 × 10(5)Da by high performance gel permeation chromatography. The structure was elucidated using methylation analysis, partial acid hydrolysis, NMR and ESI-MS analysis. Results showed that LRGP5 consisted of a (1 → 4)-linked galacturonic acid backbone occasionally interrupted by (1 → 2)-linked rhamnose. The side chains were attached to position 4 of the rhamnosyl units, including (1 → 3)-linked arabinose, (1 → 3)-linked galactose, (1 → 3,6)-linked galactose, (1 → 4)-linked galacturonic acid, (1 → 2)-linked rhamnose and (1 → 2,4)-linked rhamnose, and the termini were arabinose and rhamnose. Immunological assay results demonstrated that LRGP5 could significantly promote macrophage proliferation and enhance the secretion of nitrogen monoxide in vitro.

Sulphation pattern analysis of chemically sulphated polysaccharide LbGp1 from Lycium barbarum by GC–MS

The polysaccharide LbGp1 from Lycium barbarum L. was sulphated with sulphur trioxide-pyridine complex in DMF, yielding two sulphated polysaccharides, which were LbGp1-OL-SL with 13.7% sulphate content, and LbGp1-OL-SH with 27.4% sulphate content. The sulphation patterns were analysed using a GC-MS strategy. After a series of sequential chemical derivatisations, the sulphated polysaccharides were converted to partially methylated alditol acetates. All the sulphate groups were replaced by acetyl groups, maintaining the positional information of the original sulphation pattern. The number and position of sulphate substitutions were deduced by comparing the relative molar ratio from methylation analysis between native polysaccharide and sulphated derivatives. In LbGp1-OL-SL, 12.65% of sulphation located on C-5 of Ara, only 0.69% and 0.34% of sulphation occurred on C-4 and C-6 Gal, respectively; while in LbGp1-OL-SH, 24.96% of sulphate groups were found at C-5 of Ara, and 0.40% and 2.02% of sulphate groups were found at C-4 and C-6 Gal, respectively.

Electrospray Ionization Mass Spectrometric Analysis of κ-Carrageenan Oligosaccharides Obtained by Degradation with κ-Carrageenase from Pedobacter hainanensis

κ-Carrageenan was degraded with a novel κ-carrageenase isolated from Pedobacter hainanensis, which was first isolated from seaside soil under the stacks of red algae in Hainan province of China. The κ-carrageenase was detected with a molecular weight of ∼55 kDa estimated from SDS-PAGE and yielded enzymatic activity of 700.53 units/mg of protein under the conditions of pH 7.0 and 40 °C. Analysis of the degradation products by TLC and HPLC indicated that the enzyme degraded κ-carrageenan to sulfated oligosaccharides with even-numbered degree of polymerization, of which the tetrasaccharide was the major product. All the degradation components during different time courses were analyzed by ESI-MS, and their structures were assigned. Structural analysis by CID MS/MS revealed that each carrageenan oligosaccharide was composed of An-G4S-type neocarrabiose units, which consisted of a 3,6-anhydro-α-d-galactose (An) residue in the nonreducing end and a β-d-galactose-4-sulfate (G4S) residue in the reducing end. These results demonstrated that the κ-carrageenase cleaved κ-carrageenan at the internal β-1,4 linkage of κ-carrageenan. This enzymatic degradation offers an alternative approach to prepare κ-carrageenan oligosaccharides, which could be used as a powerful tool for further study on biological activity-structure relationship and thorough industrial exploitation of κ-carrageenan.

Comparison of chicken and pheasant ovotransferrin N-glycoforms via electrospray ionization mass spectrometry and liquid chromatography coupled with mass spectrometry.

Species-specific ovotransferrin features a highly conservative protein sequence, but it varies in the structure of the attached oligosaccharides, which may contribute to the differences observed in its bioactivity and nutritional value. Herein, chicken ovotransferrin (COT) and pheasant ovotransferrin (POT) isolated by repeated ethanol precipitation of egg white were digested with peptide N-glycosidase F to release N-glycans. The obtained N-glyans were isotopically labeled with aniline and analyzed via electrospray ionization mass spectrometry and online hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS). Relative quantitation based on isotopic aniline labeling and HILIC-MS/MS analysis revealed in detail the conspicuous difference between COT and POT in the abundance of their N-glycan compositions and isomers. In total, 16 COT N-glycans were observed, including 1 core structure (3.18%), 3 hybrid type (5.42%), and 12 complex type (91.40%), whereas 21 POT N-glycans were found, including 1 truncated structure (1.88%), 1 core structure (6.26%), 3 high mannose type (5.20%), 6 hybrid type (19.14%), and 10 complex type (67.52%). To our knowledge, this study is the first qualitative and quantitative comparison of COT and POT N-glycosylation patterns. These results suggest that POT has a different glycosylation pattern compared to that of COT and thus the effect of its glycosylation pattern on its bioactivity is worthy of further exploration.

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies.

Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d0/d5)phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis

Rapid, simple and versatile methods for quantitative analysis of glycoprotein O-glycans are urgently required for current studies on protein O-glycosylation patterns and the search for disease O-glycan biomarkers. Relative quantitation of O-glycans using stable isotope labeling followed by mass spectrometric analysis represents an ideal and promising technique. However, it is hindered by the shortage of reliable nonreductive O-glycan release methods as well as the too large or too small inconstant mass difference between the light and heavy isotope form derivatives of O-glycans, which results in difficulties during the recognition and quantitative analysis of O-glycans by mass spectrometry. Herein we report a facile and versatile O-glycan relative quantification strategy, based on an improved one-pot method that can quantitatively achieve nonreductive release and in situ chromophoric labeling of intact mucin-type O-glycans in one step. In this study, the one-pot method is optimized and applied for quantitative O-glycan release and tagging with either non-deuterated (d0-) or deuterated (d5-) 1-phenyl-3-methyl-5-pyrazolone (PMP). The obtained O-glycan derivatives feature a permanent 10-Da mass difference between the d0- and d5-PMP forms, allowing complete discrimination and comparative quantification of these isotopically labeled O-glycans by mass spectrometric techniques. Moreover, the d0- and d5-PMP derivatives of O-glycans also have a relatively high hydrophobicity as well as a strong UV adsorption, especially suitable for high-resolution separation and high-sensitivity detection by RP-HPLC-UV. We have refined the conditions for the one-pot reaction as well as the corresponding sample purification approach. The good quantitation feasibility, reliability and linearity of this strategy have been verified using bovine fetuin and porcine stomach mucin as model O-glycoproteins. Additionally, we have also successfully applied this method to the quantitative O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples.nnnBIOLOGICAL SIGNIFICANCEnO-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs.