Structural Determination Of Oligosaccharides Research Articles

Unusual free oligosaccharides in human bovine and caprine milk

Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes.

Logically derived sequence tandem mass spectrometry for structural determination of Galactose oligosaccharides.

Mass spectrometry has high sensitivity and is widely used in the identification of molecular structures, however, the structural determination of oligosaccharides through mass spectrometry is still challenging. A novel method, namely the logically derived sequence (LODES) tandem mass spectrometry (MSn), for the structural determination of underivatized oligosaccharides was developed. This method, which is based on the dissociation mechanisms, involves sequential low-energy collision-induced dissociation (CID) of sodium ion adducts, a logical sequence for identifying the structurally decisive product ions for subsequent CID, and a specially prepared disaccharide CID spectrum database. In this work, we reported the assignment of the specially prepared galactose disaccharide CID spectra. We used galactose trisaccharides and tetrasaccharides as examples to demonstrate LODES/MSn is a general method that can be used for the structural determination of hexose oligosaccharides. LODES/MSn has the potential to be extended to oligosaccharides containing other monosaccharides provided the dissociation mechanisms are understood and the corresponding disaccharide database is available.

Simple Method for De Novo Structural Determination of Underivatised Glucose Oligosaccharides

Carbohydrates have various functions in biological systems. However, the structural analysis of carbohydrates remains challenging. Most of the commonly used methods involve derivatization of carbohydrates or can only identify part of the structure. Here, we report a de novo method for completely structural identification of underivatised oligosaccharides. This method, which can provide assignments of linkages, anomeric configurations, and branch locations, entails low-energy collision-induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of four underivatised glucose oligosaccharides. Then, high-performance liquid chromatography and a mass spectrometer with a built-in logical procedure were established to demonstrate the capability of the in situ CID spectrum measurement and structural determination of the oligosaccharides in chromatogram. This consolidation provides a simple, rapid, sensitive method for the structural determination of glucose oligosaccharides, and applications to oligosaccharides containing hexoses other than glucose can be made provided the corresponding disaccharide databases are available.

High-Yielding and Controlled Dissociation of Glycosides Producing B- and C-Ion Species under Collision-Induced Dissociation MS/MS Conditions and Use in Structural Determination

Collision-induced dissociation (CID) in mass spectrometry is a powerful technique with which to understand gas-phase chemical reactions. A mass spectrometer is used to carry out the reaction, isolation, and analysis. On the other hand, structural analysis of glycan structures is of extreme importance in the analysis of biomolecules, such as glycoproteins and glycolipids. In the analysis of glycan structures based on CID, certain ion species, including B-/Y-, C-/Z-, and A-/X-ions, are produced. Among these ions, we are interested in C-ion species that carry a glycosyl oxygen atom at the anomeric center and that possibly provide information regarding anomeric configuration. A method for generating C-ion species when necessary is thus considered to be important; however, none is currently available. In this study, synthetic glycosides carrying a series of aglycons were analyzed with the aim of identifying suitable glycosides with which to produce C-ions to be used in the structural determination of oligosaccharides. The results showed a 4-aminobutyl group was an excellent candidate. Furthermore, the use of C-ion species obtained in this manner in the structural characterization of a ganglioside, GM3, is described. The type of glycoside is believed to be valuable not only in structural analysis but also in biological investigation, because of the existing amino functionality that has been proven to be useful by enabling the generation of conjugates with other molecules and materials.

Structural determination of oligosaccharides from recombinant iduronidase released with peptide N-glycanase F using fluorophore-assisted carbohydrate electrophoresis.

The lysosomal storage disorder mucopolysaccharidoses I (MPS I) is caused by a deficiency in the production of alpha-L-iduronidase. Recently, a recombinant alpha-L-iduronidase has been produced in Chinese hamster ovary (CHO) cells. It is thought that for alpha-L-iduronidase to be correctly targeted to the lysosomal vesicle a particular oligosaccharide make-up must be present, and characterization of the carbohydrates is critical. Oligosaccharides from alpha-L-iduronidase were analyzed using fluorophore-assisted carbohydrate electrophoresis (FACE). The FACE system uses polyacrylamide gel electrophoresis to separate, quantify, and determine the sequence of oligosaccharides released from glycoproteins. Asparagine-linked oligosaccharides were released from alpha-L-iduronidase using the enzyme peptide N-glycanase F (PNGase F). Released oligosaccharides were labeled with a fluorophore at the reducing termini by reductive amination. A total of nine bands were sequenced from the released pool of oligosaccharides. The pool of fluorescently labeled oligosaccharides was then electrophoresed in preparative gels and each band individually excised and extracted. Isolated bands were treated with a series of exoenzymes to determine the sequence of monosaccharides that make up a particular oligosaccharide. A total of eighteen different oligosaccharides were identified from the original pool of oligosaccharides. A majority of the oligosaccharides, over 73%, were found to be of the sialylated complex type. Four of the oligosaccharides were phosphorylated, making up approximately 11% of the carbohydrate pool, and the remaining 15% were of the oligomannose type.

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human α1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.