摘要:

AbstractThis review summarizes publications on capillary electrophoresis (CE) of carbohydrates, covering almost all hitherto published papers on this topic. It is designed to be a convenient tool for the literature search by providing a comprehensive table. Since CE analysis of carbohydrates is generally complicated due to the structural diversity of carbohydrate species, an attempt is made in this table to supply detailed information on the analyzed form (underivatized or derivatized, type of derivative) and analytical conditions (capillary size, state of the inner wall, composition of the electrophoretic solution, applied voltage, detection method,etc.), for each combination of carbohydrate species to be analyzed. In addition, a brief overview is presented to help in the literature search.

ISSN:0173-0835    

DOI:10.1002/elps.1150191503

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractProteoglycans participate and regulate several physiological processesviatheir glycosaminoglycan constituents. For a deeper understanding of how they interact with extracellular ligands as well as with cell bound effector molecules, the fine chemical structures of their glycosaminoglycan chains must be elucidated. Lately developed capillary electrophoretic techniques is a powerful analytical tool for the analysis of glycosaminoglycans, combining a high resolving power with sensitive detection. In this review we describe how depolymerized and intact glycosaminoglycans/proteoglycans can be characterized by capillary electrophoresis, relating these analyses to their possible biological significance. Conditions for running these separations and the detection systems for particular applications are also summarized.

ISSN:0173-0835    

DOI:10.1002/elps.1150191504

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractIn many ways electrophoretic techniques appear ideal for quality monitoring of proteins and are thus well suited for the analysis of recombinant glycoproteins. The requirements of high throughput, comparative analysis and resolution of many variants are met by several electrophoretic techniques. A wide variety of such techniques are available to biotechnologists in the rapidly developing area of recombinant glycoproteins. It is the aim of this review to specifically cover recent work which has been applied to the analysis of DNA‐derived glycoproteins, both from a process control standpoint and final product validation. All major areas of electrophoresis including sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), isoelectric focusing and techniques utilizing capillary electrophoresis are covered, with emphasis on analysis of glycoforms and oligosaccharide profiles of recombinant glycoproteins. As illustration, actual examples rather than standard glycoproteins are given to indicate the potential and limitations which may be encountered. It is anticipated that this review will prove a useful and practical guide to the latest developments by indicating the relevant merits of different me

ISSN:0173-0835    

DOI:10.1002/elps.1150191505

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

Abstractα‐Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex‐type oligosaccharides. Serum AFP samples from cord blood on full‐term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two‐dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above‐mentioned AFP samples. Known specificities of the lectins against complex‐type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities fo

ISSN:0173-0835    

DOI:10.1002/elps.1150191506

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore‐assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore‐assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase‐debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore‐assisted carbohydrate electrophoresis is di

ISSN:0173-0835    

DOI:10.1002/elps.1150191507

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe lysosomal storage disorder mucopolysaccharidoses I (MPS I) is caused by a deficiency in the production of α‐L‐iduronidase. Recently, a recombinant α‐L‐iduronidase has been produced in Chinese hamster ovary (CHO) cells. It is thought that for α‐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 α‐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 α‐L‐iduronidase using the enzyme peptideN‐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

ISSN:0173-0835    

DOI:10.1002/elps.1150191508

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteriaBradyrhizobium (Chamaecytisus)strain BGA1 andBradyrhizobium japonicumUSDA110 has been studied. Both polysaccharides were polydisperse, producing a ladder‐like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced byB. japonicumUSDA110 was wider and with larger molecules than that of the polysaccharide produced by strain BGA1. The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris‐borate; in Tris‐glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate

ISSN:0173-0835    

DOI:10.1002/elps.1150191509

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractCapillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection has been used for the determination of glucose in samples collected by noninvasive means. The method uses an enzymatic reaction scheme that provides for the determination of small quantities of glucose with detection limits of 80 nM. This approach is used to evaluate passive transdermal diffusion as a noninvasive means to sample glucosein vivo.A simple sampling cell design is presented. Sample collection was performed on volunteer human subjects. Our experiments show that fluctuations in blood glucose concentration are reflected in the samples obtained by passive transdermal diffusion after glucose intake. The results indicate that glucose from the subcutaneous fluid can be accessed by passive diffusio

ISSN:0173-0835    

DOI:10.1002/elps.1150191510

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractOligomers of glucose and oligosaccharides released from glycoproteins were derivatized with 2‐aminobenzamide. As this fluorophore imparts no charge to the oligosaccharides, several strategies were investigated to achieve capillary electrophoresis (CE) separation of both neutral and charged derivatized glycans. Micellar electrokinetic capillary chromatography (MEKC) with the addition of anionic surfactants was evaluated as a first approach: sodium dodecyl sulfate (SDS) produced the best separation of the oligoglucose fragments, where the migration was inversely related to their degree of polymerization. To demonstrate the applicability of this method for complex carbohydrate analysis, oligosaccharide mixtures derived from ribonuclease B (RNase B) and α‐acid glycoprotein (α‐AGP) were analyzed. A satisfactory separation for the high‐mannose structures found in RNase B could be obtained, whereas charged oligosaccharides from α‐AGP were poorly resolved. Cyclodextrin‐modified CE was chosen as the second approach: the effect of the addition of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) or sulfobutylether‐γ‐cyclodextrin (SBE‐γ‐CD) on the electrophoretic mobilities and resolution of neutral and charged oligosaccharides was then studied. Selectivity of sialylated structures could be further improved by using anionic cyclodextrins (CDs) instead of micelles. However, this latter approach failed to baseline‐resolve the different high‐mannose structures of RNase B. A successful separation of the complex mixture of oligosaccharides from α‐AGP was obtained with the addition of 4% of SBE‐γ‐CD and tri

ISSN:0173-0835    

DOI:10.1002/elps.1150191511

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA method for analysis ofN‐linked oligosaccharides derived from glycoproteins including sialic acid‐containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser‐induced fluorescence (LIF) detection. Glycoproteins were heat‐denatured in the presence of a reducing agent and theN‐linked oligosaccharides were released by peptideN‐glycosidase (PNGase F; EC3.5.1.52)‐catalyzed hydrolysis. The releasedN‐linked oligosaccharides were derivatized with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A modelN‐linked oligosaccharide, desialylated, galactosylated biantennary, core‐substituted with fucose (A2F) was tested for APTS‐based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylatedN‐linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resoluton of theN‐linked oligosaccharide profile for fingerprinting gly

ISSN:0173-0835    

DOI:10.1002/elps.1150191512

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY