Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-10
2001-09-25
Allen, Marianne P. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S123000, C536S025400, C204S456000, C204S459000, C204S461000, C204S548000, C436S172000
Reexamination Certificate
active
06294667
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns the analysis of carbohydrates
BACKGROUND TO THE INVENTION
The publication by Wenn (Wenn, R. V. (1975) Biochem. J. 145, 281-285) discloses, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances, involving labelling glycopeptides with a fluorescent labelling reagent, e.g. dansylcadaverine, and separating them by electrophoresis on a paper matrix (paper-electrophoresis) after application to that matrix. The fluorescent labelling reagent imparts a charge to the carbohydrates enabling their electrophoretic separation and it also enables the visualisation of the substances after the electrophoresis.
The publication by West (West, M. H. P., et al. (1984) Electrophoresis 3, 133-138) discloses a technique for the electrophoretic separation of charged substances of relatively low molecular weight, such as amino acids and their oligomers, involving applying the amino acids to an electrophoretic gel made of a polymer of acrylamide, that is a polyacrylamide gel, and separating the substances by the application of an electrical potential to the gel. This technique is known as PAGE. The publication by West also discloses that an electrophoretic gel matrix of relatively high concentration of polyacrylamide enables the separation of the amino acid leucine from its dimer, leucylleucine which in turn is separated from the homologous trimer, leucylleucylleucine and so on. In the publication by West the amino acids were rendered detectable by the use of amino acids that comprised radioisotopic atoms in their structures.
The publication of Weitzman et al. (Weitzman, S., et al. (1979) Anal. Biochem. 97,438-439.) discloses that carbohydrate substances of relatively low molecular weight can be separated by electrophoresis in a polyacrylamide gel when an electrophoretic buffer system that contains borate ions is used.
The publication by Poretz and Pieczenik (Poretz, R. D. and Pieczenik, G. (1981) Anal. Biochem. 115, 170-176) discloses the labelling with a fluorescent labelling reagent of glycopeptides and also discloses their separation after electrophoresis in a polyacrylamide gel and their detection by the incorporated fluorescent label.
The publication by Prakash and Vijay (Prakash, C. and Vijay, I. A. (1983) Anal. Biochem. 128, 41-46) discloses the labelling of carbohydrate substances with a fluorescent labelling reagent.
The publication by Towbin et al. (Towbin, H., et al. (1988) Anal. Biochem. 173, 1-9) discloses the labelling of carbohydrate substances with a chromophore.
The publication by Hase et al. (Hase, S., et al. (1978) J. Biochem. 85, 989-994) discloses the electrophoretic separation by paper electrophoresis in two dimensions of carbohydrate substances labelled with the fluorescent labelling reagent aminopyridine.
The publications by Jackson (Jackson P., (1990) Biochem. J, 270, 705-713 and Jackson, P. 1992) Anal. Biochem. 196, 238-244) and international publications No. WO88/10422, No. WO91/05256, No. WO93/02356, and No. WO92/11531, disclose, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances involving applying carbohydrate substances to an electrophoretic gel to cause differential migration of different substances. The carbohydrate substances may be pre-labelled with a fluorescent labelling reagent, for example 8-aminonaphthalene-1,3,6-trisulphonic acid (known as ANTS for brevity) to impart a charge to the substances, thereby to enable electrophoretic separation, and to enable visualisation of the substances after running the gel. Visualisation may be effected by the naked eye but other means such as photography or electronic imaging may also be used.
The techniques described as examples in the patents which are cited above involve the use of negatively charged or uncharged fluorescent labelling reagents. The separations of fluorescently labelled carbohydrate substances that have been described so far show a relatively limited degree of separation of carbohydrate substances a significant proportion of which are not resolved from each other by the electrophoresis in a useful way. In particular this applies to the asparagine linked glycans that have been analysed as shown in the publication by Stack and Sullivan (Stack, R. J., and Sullivan, M. T. (1992) Glycobiology 2, 85-92). The invention described herein is a novel and innovative development that is related to the methods that are described in the papers and patents cited above.
BRIEF SUMMARY OF THE INVENTION
The subject of the invention is the separation of the fluorescently labelled carbohydrate substances, by virtue of their different charge-to-mass ratios or other factors, so as to enable a much larger number of different fluorescently labelled carbohydrate substances to be separated from each other electrophoretically than has been possible previously and thereby to facilitate their structural determination and their identification. The invention is aimed in particular, but not exclusively, at resolving carbohydrate substances that have been released from glycoproteins, proteoglycans and glycolipids. Some of the procedures that are described do not involve electrophoretic separation but are related to it and provide for additional analyses or identification of the carbohydrate substances.
Preferably the method for separating or distinguishing carbohydrate substances comprises labelling carbohydrate substances with a fluorescent labelling reagent comprising a naphthalene ring structure or other suitable fluorescent structure, having as a substituent a reactive group capable of reacting with a reducing sugar to bind thereto, also having at least one substituent, that may also be the reactive group, capable of carrying at least one positive charge which may exist on the fluorescently labelled carbohydrate substances and does not extinguish the fluorescence of the labelling reagent; applying the labelled substances to an electrophoretic gel, or other matrix used to support electrophoretic separations, and running the electrophoresis to cause differential migration of different substances. The labelling reagent may be attached to sites on the carbohydrate substances after release, if necessary, from an attached biomolecule. Alternatively, the biomolecule may be modified in known ways to enable incorporation of the labelling reagent. A carbohydrate substance may be labelled with a labelling reagent such as those indicated in Table 1 by incubating the substances with the labelling reagent. In the case of some labelling reagents, such as those reacting by way of an amino group, it is preferable to add a reducing agent (e.g. sodium cyanoborohydride). The sodium cyanoborohydride is conveniently in solution in dimethylsulphoxide or other suitable solvent known to those skilled in the art.
REFERENCES:
patent: 5268486 (1993-12-01), Waggoner et al.
patent: 5284558 (1994-02-01), Linhardt et al.
patent: 5453505 (1995-09-01), Lee et al.
patent: 5472582 (1995-12-01), Jackson
patent: 3912046 (1990-03-01), None
patent: 0 233 053 (1987-08-01), None
patent: WO 88/10422 (1988-12-01), None
patent: WO 91/05256 (1991-04-01), None
patent: WO 91/11531 (1992-07-01), None
patent: WO 93/02356 (1993-02-01), None
patent: WO 96/00902 (1996-01-01), None
patent: WO 96/04405 (1996-02-01), None
patent: WO 96/13552 (1996-05-01), None
Hase, S. et al., “Analyses of Oligosaccharides by Tagging the Reducing End with a Flourescent Compound,”J. Biochem., 85(4):989-994 (1979).
Jackson, P. et al., “The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid,”Biochem. J., 270:705-713 (1990).
Jackson, P., “Polyacrylamide Gel Electrophoresis of Reducing Saccharides Labeled with the Fluorophore 2-Aminoacridone: Subpicomolar Detection Using an Imaging System Based on a Cooled Charge-Coupled Device,”Analytical Biochemistry, 196:238-244 (1991).
Briggs Mark Samuel Jonathan
Cummins William Jonathan
Jackson Peter
Smith John Anthony
West Richard
Allen Marianne P.
Amersham International plc
Marshall O'Toole Gerstein Murray & Borun
Moran Marjorie A
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