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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, enormous multisystemic disorders,

Inactive Publication Date: 2006-03-16
MOMENTA PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0033] According to another aspect of the invention, methods of generating a list of glycoconjugate properties are provided. The methods include measuring two or more properties of the glycoconjugate, and recording a value for the two or more properties of the glycoconjugate to generate a list, wherein the value of the two or more properties is recorded in a computer-generated data structure. In some embodiments, one of the two or more properties of the glycoconjugates is the number of one or more types of monosaccharides of the glycoconjugate. In other embodiments, one of the two or more properties of the glycoconjugates is the total mass of the glycans of the glycoconjugate. In still other embodiments, the glycoconjugate is a glycoprotein or proteoglycan, and one of the two or more properties of the glycoconjugate is the mass of the peptide of the glyco

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., Takamiya, K., Okada, M., Inoue, M., Fukumoto, S.

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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Effect test

example 1

N-Glycan Analysis

Materials and Methods

PNGaseF Digest of N-Glycans from Protein Cores

[0153] Between 10 and 100 μg of protein was 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 50 mM sodium phosphate buffer, pH 7.5.

Purification of Released N-Glycans

[0154] 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.

[0155] 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 1 references

[0181] 1. Hirschberg, C. B., Snider, M. D. (1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 56, 63-87. [0182] 2. Bause, E. (1983) Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331-6. [0183] 3. Marshall, R. D. (1972) Glycoproteins. Annu Rev Biochem 41, 673-702. [0184] 4. Dwek, R. A. (1996) Glycobiology: Toward Understanding the Function of Sugars. Chem Rev 96, 683-720. [0185] 5. O'Connor, S. E., Imperiali, B. (1996) Modulation of protein structure and function by asparagine-linked glycosylation. Chem Biol 3, 803-12. [0186] 6. Crocker, P. R., Varki, A. (2001) Siglecs in the immune system. Immunology 103, 137-45. [0187] 7. Helenius, A., Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364-9. [0188] 8. Imperiali, B., O'Connor, S. E. (1999) Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opi...

example 2

Profiling of N-Glycans from Human Serum

Materials and Methods

Cleavage of N-Glycans from Serum Glycoproteins (Reduction / Carboxymethylation Method)

[0217] 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 MWCO filter. 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

[0218] After glycans were cleaved from the protein, the sample was...

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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. Improved methods, therefore, for the study of glycosylation patterns on cells, tissue and body fluids are also provided. Information regarding 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 on protein function. Such methods are also provided. Methods are also provided to assess protein production processes, to assess the purity of proteins produced, and to select proteins with the desired glycosylation.

Description

RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 from U.S. provisional application Ser. No. 60 / 562,874, filed Apr. 15, 2004, the entire contents of which is 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 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 fluid, such as serum, are also provided. Information regarding the glycosylation patterns on cells can be used in diagnostic and treatment methods a...

Claims

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Application Information

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IPC IPC(8): G01N33/53G06F19/00G01N33/68
CPCG01N33/6803G01N33/6842Y10T436/143333G01N2400/10G01N33/6851
Inventor BOSQUES, CARLOSKEISER, NISHLASRINIVASAN, ARAVINDSASISEKHARAN, RAMGANDHE, PANKAJRAGURAM, SASI
Owner MOMENTA PHARMA
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