The Determination of Carbohydrate in Biological Materials by Gas‐Liquid Chromatography

Clamp, John R.; Bhatti, Tariq M.; Chambers, Robin E.

doi:10.1002/9780470110386.ch3

Cited by 207 publications

(2 citation statements)

References 312 publications

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“…Trimethylsilylation of hydroxyl and carboxyl groups is the most widely used method. 267,268 If only neutral monosaccharides need to be measured, acid hydrolysis followed by reduction and peracetylation (alditol acetate derivatives) is preferred. 269,270 …”

Section: Gas -Liquid Chromatographic Analysis Of Monosaccharidesmentioning

confidence: 99%

Glycoprotein Analysis: General Methods

Debray¹,

Spik²

2000

Encyclopedia of Analytical Chemistry

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Glycosylation is a common co‐ and post‐translational modification of a protein which may have profound effects on protein structure and function. Many biologically interesting proteins are glycosylated at their asparagine, serine, and threonine residues. Glycosylation has now been recognized as being more ubiquitous and structurally varied than all other types of post‐translational modifications combined. The glycosylation pattern of a glycoprotein is not random but is dependent on cell type, physiological conditions and, in some cases, disease states. Carbohydrate chains or glycans attached to the peptide backbone are classified according to the nature of the linkage to the peptide and their structure; both of these parameters allow the definition of common cores. In addition to the extreme structural diversity and complexity of glycan structures, one feature of protein glycosylation is the presence at a single site of any one of a number of glycan structures. This property gives rise to an extremely heterogeneous population of glycoproteins, termed glycoforms. The complexity of the glycan structures, the multiple substitutions (microheterogeneity) at glycosylation sites, and the structural diversity associated with the protein backbone itself represent an enormous task for analytical structural studies. Indirect methods such as immunological approaches using antibodies or lectins provide some structural information concerning the nature of glycans. Prior to their structural study, glycans have to be released from the protein backbone by enzymatic or chemical methods. Different endoenzymes such as N ‐glycanase ® allow a complete deglycosylation of glycoproteins, providing a pool of oligosaccharides which may be further fractionated. Establishment of the site heterogeneity and assessment of the different glycoforms require isolation and analysis of the individual glycopeptides generated after hydrolysis with specific proteases. Fractionation of glycans or glycopeptides can be achieved by different high‐performance liquid chromatography (HPLC) or electrophoretic procedures. Lectins, due to their extreme specificities with regard to monosaccharide or oligosaccharide motifs, represent powerful tools for sequential glycan fractionation. Traditionally, a complete structural analysis of the glycan structures, including determination of the carbohydrate sequence and the sugar linkage, has been a tedious, multidisciplinary task. Characterization and determination of the relative amount of individual constituent monosaccharides is generally obtained by gas–liquid chromatography (GLC) after hydrolysis of the oligosaccharide chain. Information concerning the nature of linkages and branching points is furnished by the methylation procedure. Nevertheless, these chemical sequencing methods often necessitate milligrams of material. To progress from this situation, sensitive physicochemical or enzymatic methods have been developed. The nuclear magnetic resonance (NMR) study of oligosaccharides, even of the less sensitive of the physicochemical techniques, is extremely powerful because it allows determination in a mixture of glycans, the complete structure of individual glycans including nature of monosaccharides, sequence, type of linkages, anomericity and branching points. The recent advances in mass spectrometry (MS) techniques, mainly matrix‐assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI), have provided very sensitive means for the analysis of oligosaccharides and glycopeptides. The gain in sensitivity associated with a minimum number of sample manipulation steps allows work at the submicrogram level. The use of enzyme reagents is rapidly becoming popular in modern carbohydrate analysis because of their inherent selectivities for a sugar substrate and its linkage type. Exoglycosidases can be used in conjunction with other methods such as MALDI/MS or fluorophore‐assisted carbohydrate electrophoresis (FACE) separation. Complete structural information is now feasible for low‐microgram and submicrogram quantities of a glycoprotein; this quality of performance places the field of carbohydrate analysis closer to the sequencing methods available for other biomolecules, proteins or nucleic acids.

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Section: Gas -Liquid Chromatographic Analysis Of Monosaccharidesmentioning

confidence: 99%

Glycoprotein Analysis: General Methods

Debray¹,

Spik²

2000

Encyclopedia of Analytical Chemistry

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“…Carbohydrates were analyzed as previously described for purified normal liver a-L-fucosidase (Alhadeff & Freeze 1977) by gas-liquid chromato-graphy (glc) using trimethylsilyl (TMS) derivatives of the methyl glycosides, essentially as described by Etchison & Holland (1974). Samples containing 140-300 pg of enzyme protein (Lowry) were lyophilized in screw-cap vials containing 5 pg of arabitol (internal standard) and the samples were methanolyzed with 0.5 ml of 1.5 N anhydrous methanolic-HC1, prepared according to Clamp et al (1973). Analyses were performed on a Hewlett-Packard 5700A gas chromatograph, equipped with dual 6' x %'' glass columns and an all-glass line to the flame ionization detector.…”

Section: Piirification Of Cystic Fibrosis Liver A-l-mentioning

confidence: 99%

Purification and characterization of altered cystic fibrosis liver α‐L‐fucosidase

1978

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α‐L‐Fucosidase (E.C.3.2.1.51) from two cystic fibrosis livers has been purified and characterized. Purification was accomplished by an affinity chromatographic procedure previously used for normal liver α‐L‐fucosidase. Characterization of the two cystic fibrosis α‐L‐fucosidases indicated that they were very similar to normal liver α‐L‐fucosidase with regard to pH optima profiles, Michaelis constants (Km's), subunit structure and antigenicity. However, gas liquid chromatographic analysis revealed altered carbohydrate compositions for both the cystic fibrosis α‐L‐fucosidases. The three major sugars found in normal purified liver α‐L‐fucosidase (mannose, N‐acetylglucosamine and sialic acid) were reduced in the cystic fibrosis α‐L‐fucosidases, on average, to 51 %, 44 % and 32 %, respectively, of their normal amounts,

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Phlyctenulae and Catarrhal Infiltrates

Mondino¹

1982

Arch Ophthalmol

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The Determination of Carbohydrate in Biological Materials by Gas‐Liquid Chromatography

Cited by 207 publications

References 312 publications

Glycoprotein Analysis: General Methods

Glycoprotein Analysis: General Methods

Purification and characterization of altered cystic fibrosis liver α‐L‐fucosidase

Phlyctenulae and Catarrhal Infiltrates

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