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Methods and products related to the improved analysis of carbohydrates

a technology of improved analysis and carbohydrates, applied in the field of improved analysis of carbohydrates, can solve the problems of embryonic lethality and enormous multisystemi

Inactive Publication Date: 2006-06-15
MASSACHUSETTS INST OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0074] In still another aspect of the invention a method of creating a database of glycoprofiles, which includes generating a glycoprofile of a sample according to a method provided, and recording one or more values corresponding to the glycoprofile in a computer-generated data structure is provided. In yet another aspect of the invention the database so created is also provided.
[0075] In another aspect of the invention a method of determining a glycome pattern, which includes obtaining a glycoprofile of total carbohydrates (e.g., glycans) of a sample with a method provided herein, identifying features of the glycoprofile, generating data sets based on the features of the glycoprofile, identifying a pattern in the data sets, and determining whether or not the pattern is associated with a known sample or diseased state is provided. In one embodiment the sample is obtained from a subject. In another embodiment the subject has a disease or condition. In another embodiment determining the glycoprofile includes obtaining more than one glycoprofile spectra. In one embodiment one of the spectra is of acidic carbohydrates (e.g., glycans). In another embodiment one of the spectra is of neutral carbohydrates (e.g., glycans). In still another embodiment one spectra is of acidic carbohydrates (e.g., glycans) and another spectra is of neutral carbohydrates (e.g., glycans).
[0076] In yet another embodiment, when the analysis, includes the generation of one or more glycoprofile spectra, the methods provided can also include assigning all of the possible carbohydrates (e.g., glycans) to the peaks of the one or more spectra.
[0077] In one embodiment the feature identified is the presence of one or more carbohydrates (e.g., glycans), the absence of one or more carbohydrates (e.g., glycans), the relative amount of one or more carbohydrates (e.g., glycans), the combination of two or more classes of carbohydrates (e.g., glycans), the presence of a specific carbohydrate (e.g., glycan) motif (i.e., a specific set of one or more monomers (e.g., monosaccharides)), the absence of a specific carbohydrate (e.g., glycan) motif, the relative amount of a specific carbohydrate (e.g., glycan) motif, the presence of one or more monomers in a carbohydrate (e.g., glycan), the absence of one or more monomers in a carbohydrate (e.g., glycan), the relative amount of one or more monomers in a carbohydrate (e.g., glycan) or the bond between monomers of a carbohyrdate.

Problems solved by technology

In mouse knockout models, disrupting even one of the biosynthetic enzymes can lead to enormous multisystemic disorders, and several result in embryonic lethality (Furukawa, K., et al.

Method used

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  • Methods and products related to the improved analysis of carbohydrates
  • Methods and products related to the improved analysis of carbohydrates
  • Methods and products related to the improved analysis of carbohydrates

Examples

Experimental program
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example 1

N-Glycan Analysis

Materials and Methods

PNGaseF Digest of N-Glycans from Protein Cores

[0219] Between 10 and 100 μg of protein were denatured for 10 minutes at 90° C. with 0.5% SDS and 1% β-mercaptoethanol. Since SDS (and other ionic detergents) inhibits enzyme activity, 1% NP-40 was added to counteract these effects. The enzyme reaction was performed overnight with 2 μl of PNGaseF at 37° C. in a 50mM sodium phosphate buffer, pH 7.5.

Purification of Released N-Glycans

[0220] Proteins were precipitated with a 3× volume of 100% ethanol on ice for 1 hour. After centrifugation to remove the proteins, the supernatant containing the N-glycans was evaporated by vacuum (SpeedVac, TeleChem International, Inc., Sunnyvale, Calif.). Dried glycans were resuspended in 50 μl of water.

[0221] Samples were desalted using 1 ml ion exchange column of AG50W X-8 beads (Bio-Rad, Hercules, Calif.). The resin was charged with 150 mM acetic acid and washed with water. Glycan samples were loaded onto the ...

example 2

Profiling of N-Glycans from Human Serum

Materials and Methods

[0246] Cleavage of N-glycans from Serum Glycoproteins (Reduction / Carboxymethylation Method)

[0247] Human male normal serum samples were obtained from IMPATH (Franklin, Mass.) and Biomedical Resources (Hatboro, Pa.), and stored at −85° C. For each experiment, 50 μl of serum was used to harvest N-glycans. Serum samples were first diluted 1:4 with water, then DTT was added to a final concentration of 80 mM. After incubation for 30 minutes at 37° C., iodoacetic acid was added to a final concentration of 400 mM and incubated for 1 hour more at 37° C. The sample was dialyzed against 10 mM Tris acetate pH 8.3 overnight and concentrated to ˜200 μl in a spin column with a 3000 Da Molecular Weight Cut off (MWCO) filter (VivaScience, Hannover, Germany). To cleave the sugars from the protein, 5 μl (1,000 U) of PNGaseF (New England Biolabs, Beverly, Mass.) was added and allowed to react overnight at 37° C.

Purification of N-Glycans ...

example 3

Glycan Analysis

Release of Glycans from Proteins

[0284] Several methods were used to cleave the carbohydrates from proteins:

[0285]

[0286] A) Glycoproteins were denatured with 0.5% SDS and 1% β-mercaptoethanol. Since SDS (and other ionic detergents) inhibits enzyme activity, 1% NP-40 was added to counteract these effects. The enzymatic cleavage was performed overnight with PNGaseF (New England Biolabs) at 37° C. in sodium phosphate buffer, pH 7.5 or Tris acetate buffer pH 8.3.

[0287] B) Samples were reduced with DTT followed by alkylation with either iodoacetic acid or iodoacetamide. The sample was dialyzed against phosphate buffer, pH 7.5 or Tris acetate, pH 8.3 overnight and concentrated to ˜200 μl in a spin column with a 3000 Da MWCO filter. To cleave the sugars from the protein between 100 and 2,000 U of PNGaseF (New England Biolabs) were used.

[0288] C) Glycoproteins were denatured using a buffer containing 8M urea, 3.2 mM EDTA and 360 mM Tris, pH 8.6 (Papac, D. I., et al. 1998...

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Abstract

The invention relates, in part, to the improved analysis of carbohydrates. In particular, the invention relates to the analysis of carbohydrates, such as N-glycans and O-glycans found on proteins and saccharides attached to lipids. Improved methods, therefore, for the study of glycosylation patterns on cells, tissue and body fluids are also provided. Information from the analysis of glycans, such as the glycosylation patterns on cells, tissues and in body fluids, can be used in diagnostic and treatment methods as well as for facilitating the study of the effects of glycosylation / altered glycosylation. Such methods are also provided. Methods are further provided to assess production processes, to assess the purity of samples containing glycoconjugates, and to select glycoconjugates with the desired glycosylation.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 11 / 107,982, filed Apr. 15, 2005, which claims the benefit under 35 U.S.C. §119 from U.S. provisional application Ser. No. 60 / 562,874, filed Apr. 15, 2004. The entire contents of each of which are herein incorporated by reference.GOVERNMENT SUPPORT [0002] Aspects of the invention may have been made using funding from National Institutes of Health Grant number GM 57073. Accordingly, the Government may have rights in the invention.FIELD OF THE INVENTION [0003] The invention relates to the improved analysis of carbohydrates. In particular, the invention relates to the analysis of carbohydrates, such as N-glycans and O-glycans found on proteins and saccharides attached to lipids. The invention also relates to the analysis of glycoconjugates, such as glycoproteins, glycolipids and proteoglycans. Methods for the study of glycosylation patterns on cells, tissues and in body fluids, suc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/10G01N33/53C12P21/06G06F19/00
CPCG01N33/66G01N33/68G01N33/6842G01N33/6848G01N33/6851G01N2400/10G06F19/703G16C20/20
Inventor BOSQUES, CARLOSKEISER, NISHLASRINIVASAN, ARAVINDRAMAN, RAHULVISWANATHAN, KARTHIKSASISEKHARAN, RAMGANDHE, PANKAJRAGURAM, SASI
Owner MASSACHUSETTS INST OF TECH
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