Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis

For most separations-based analyses of glycoprotein oligosaccharides, the first step is release of the oligosaccharides from the polypeptide. Historically, O-linked and N-linked oligosaccharides have been released from glycoproteins using chemical means, such as alkaline degradation (β-elimination) or hydrazinolysis. In the last two decades, a growing repertoire of enzymes, including endoglycosidases and glycoamidases, able to release glycoprotein oligosaccharides under mild conditions, have become available. This review traces the discovery characterization and use of these glycoprotein oligosaccharide releasing enzymes. Emphasis is placed on providing information of practical value for the researcher wishing to incorporate enzymatic oligosaccharide release into their study of glycoprotein oligosaccharide structure and function.     

Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins

A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH₃CO₂]⁻ using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M − H]⁻. The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 μg of fetuin, ribonuclease B, peroxidase, and α ₁-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents.     

Enzymatic digestion and chromatographic analysis of arsenic species released from proteins

A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAs^III or MMA^III during the protease digestion process. Up to 61% DMA^III (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMA^V). The arsenic species in the red blood cells of control rats was present as DMA^III complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity.     

High performance liquid chromatographic strategy for the analysis of exopolysaccharides extracted from pathogenic bacteria.

A high performance liquid chromatographic (HPLC) method has been developed to permit the rapid comparison of acidic polysaccharides of diverse compositions and the sensitive determination of their constituents. It is based on two combined analyses of the polysaccharide hydrolysates--a separation of the released compounds by ion-moderated partition chromatography with UV detection at two wavelengths and a separation of the sugar dansylhydrazine derivatives by reversed phase chromatography. The former permits identification and quantitation of uronic and carboxylic acids, the latter permits more sensitive and specific determination of the neutral aldoses. Some bacterial exopolysaccharides have been used to demonstrate the validity of this HPLC procedure for the chemical characterization of uronic acid-containing polysaccharides. This method appears to be useful for studying capsular polysaccharides, which are involved in the evasion of phagocytosis by pathogenic bacteria.     

Compositional monosaccharide analysis of transgenic corn glycoproteins by HPLC with fluorescence detection and LC-MS with sonic spray ionization

Transgenic corn offers an attractive, cost-effective means for the large-scale production of engineered glycoproteins suitable for pharmaceutical purposes. A glycoprotein expressed in transgenic corn theoretically should not contain glycans because glycosylation sites have been genetically altered. A sensitive and reliable analytical method is developed to investigate this particular protein for the presence of glycans by monitoring the monosaccharide composition. Identification and quantitation of low-level monosaccharides in the glycoprotein hydrolyzate are accomplished by derivatization prior to high-performance liquid chromatography (HPLC)-fluorescence and liquid chromatography (LC)-sonic spray ionization (SSI)-mass spectrometry (MS) analyses. LC-SSI-MS is used to confirm the results from HPLC-fluorescence analysis and to positively identify the compositional monosaccharides. Glucosamine, glucose, mannose, arabinose, xylose, and sialic acid are found in the transgenic corn derived glycoprotein at less than one moiety per protein, which indicates heterogeneity of the particular glycoprotein. In addition to the compositional analysis of low-level monosaccharides in glycoprotein by HPLC-fluorescence, the utility of SSI for the LC-MS analysis of derivatized monosaccharides is demonstrated in this paper.     

A new monosaccharide from Swertia punicea Hemsl

A new compound named 1-hydrate,3-deoxy-α-D-tagatofuranose was isolated from Swertia punicea Hemsl.The structure of compound was determined by 1D and 2D NMR,HRESIMS techniques.     

A micropreparative chromatographic strategy for the purification and sequence analysis of murine granulocyte-colony stimulating factor (mG-CSF)

Summary A micropreparative chromatographic strategy using short (<5 cm) 1 and 2 mm ID HPLC columns for preparing pure mG-CSF and its peptide fragments is described. The proteins and peptides are used to obtain both N-terminal and internal amino acid sequence data for the purpose of constructing sequence specific oligonucleotide probes for use in the isolation of the gene coding for the protein of interest, thereby ultimately enabling amplified amounts of protein to be generated using prokaryotic expression systems.     

A new lipophilic monosaccharide from Erigeron annggs

A new lipophilic monosaccharide,erigearide A(1),was isolated from the aerial parts of Erigeron annuus(Lima.)Pers.Its structure was elucidated by analysis of spectroscopic evidence.     

A new lipophilic monosaccharide from Erigeron annuus

A new lipophilic monosaccharide, erigearide A （1）, was isolated from the aerial parts of Erigeron annuus （Lima.） Pers. Its structure was elucidated by analysis of spectroscopic evidence.     

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.     

A strategy for the identification of site-specific glycosylation in glycoproteins using MALDI TOF MS

A strategy for investigation of site-specific glycosylation of glycoproteins has been developed, based on peptide mass fingerprinting using matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF MS). The glycoprotein is subjected to sequential digestion with a protease and glycan-specific endoglycosidases or with the glycan-specific endoglycosidases followed by the protease. Peptides with characteristic masses are detected for sequences containing glycosylated asparagine residues. By using a panel of three proteases, chymotrypsin, protease V8 and trypsin, and endoglycosidases F3 and H and peptide N-glycanase F, it was possible to monitor the state of glycosylation of all putative N-glycosylation sites on three glycoproteins. It was deduced that all potential N-glycosylation sites in human serum transferrin (two) and α1-antitrypsin (three) were occupied by non-fucosylated, biantennary, disialylated, complex glycans. In contrast, only four (asparagines 19, 59, 146 and 270) out of the five potential sites were glycosylated in recombinant human β-glucosylceramidase, with the site nearest the C-terminal (asparagine 462) being unoccupied. The glycans at each site consisted of a mixture of non-fucosylated and core α1–6 fucosylated oligomannose glycans (Man₃ GlcNAc₂), derived from the enzymic truncation of complex glycans.     

A gas chromatographic analysis for SO2 oxidation

Summary An improved method of determining yields of sulphur trioxide in sulphur dioxide oxidations is reported. The method relies on the absorption of SO₃ from the reaction gases. An all-teflon system incorporating a gas density balance is used. Separation of SO₂ and O₂ is effected on a Porapak PS column at ambient temperature.     

An alternative strategy based on ultra-high-performance supercritical fluid chromatography for full monosaccharide compositional analysis of polysaccharides in Schisandra chinensis fruits

Due to green and environment-friendly characteristics, ultra-high-performance supercritical fluid chromatography has been widely used in analytical fields in recent years, but until now few reports are available for monosaccharide compositional analysis of macromolecule polysaccharides. In this study, an ultra-high-performance supercritical fluid chromatography technology with an unusual binary modifier is used to determine the monosaccharide compositions of natural polysaccharides. Each carbohydrate herein is simultaneously labeled as 1-pheny-3-methyl-5-pyrazolone and acetyl-derivative via pre-column derivatizations aiming to increase UV absorption sensitivity and decrease water solubility. Ten common monosaccharides are fully separated and detected on ultra-high-performance supercritical fluid chromatography combined with a photo-diode array detector by systematic optimization of multiple relevant parameters, for example, column stationary phases, organic modifiers, additives, flow rates, and so on. Compared with carbon dioxide as a mobile phase, the addition of a binary modifier increases the resolution of analytes. Additionally, this method has the advantages of small consumption of organic solvent, safety, and being environmental-friendly. It has been successfully applied for full monosaccharide compositional analysis of heteropolysaccharides from Schisandra chinensis fruits. To sum up, a new alternative approach is provided for monosaccharide compositional analysis of natural polysaccharides.     

Capillary liquid chromatographic fingerprint used for discrimination of Zingiber montanum from related species

Fingerprint analysis using capillary liquid chromatography (CLC) has been developed for discrimination of Zingiber montanum (ZM) from related species, for example Z. americans (ZA) and Z. zerumbet (ZZ). By comparing the fingerprint chromatograms of ZM, ZA, and ZZ we could identify ZM samples and discriminate them from ZA and ZZ by using their marker peaks. We also combined CLC fingerprint with multivariate analysis, including principal-component analysis (PCA) and canonical variate analysis (CVA); all three species were discriminated successfully. This result indicates that CLC fingerprint analysis in combination with PCA and CVA can be used for discrimination of ZM samples from samples of related species.