glycotechnology - overview a krishtech solutions presentation

Glycotechnology - Overview

A KRISHTECH SOLUTIONS Presentation

Glycotechnology - Overview


Ludger Limited are specialists on glycosylation for therapeutic proteins & pioneers in the industry.

Ludger Limited is based in UK. As their exclusive partner in India, we offer their products & services.

Our Credentials :

We are India’s only and leading player working on glycosylation. India’s first Monoclonal Antibody (MAb) launched by a leading pharmaceutical company has been glycosylated using our tools.

Today, we work with a majority of the Indian companies for glycosylating their products. Talk to us as we have the requisite experience to deliver you effective solutions for your products.

Glycan Release - Hydrazinolysis or Endoglycosidase

Glycoprotein

Hydrazinolysis

Hydrazine hydrolysis has been found to be effective in the complete release of unreduced O- and N-linked oligosaccharides. It involves the incubation of the dried glycoprotein with anhydrous hydrazine and further workup to purify the released glycans. Hydrazinolysis is a very versatile procedure and has the ability to release both N- and O-linked glycans in a virtually non-selective way.

Endoglycosidase Treatment

An alternate to hydrazinolysis is endoglycosidase release which is suitable for many applications. There are a number of useful endoglycosidases including PNGase F which releases most N-glycans from glycoproteins. When using endoglycosidases ensure suitability as there are a number of conditions and substances that can lead to selective non-release of glycans. In particular, PNGase F does not release certain types of N-glycan which bear core fucose and it can also have low activity against glycans which are found at positions near to either the C- or N-terminii of the peptide backbone.

Talk to us for glycan release – deglycosylation kits ….. Talk to us for glycan release – deglycosylation kits …..


Post Release Clean Up

Free Glycans

Post release or post deglycosylation, cleanup of glycans after enzyme digests is necessary for removal of a variety of non-carbohydrate contaminants from glycan samples including most salts and detergents used in enzymatic digests.

LudgerClean E10 glycan purification cartridges from Ludger Ltd., UK are used to clean-up free glycans.. These cartridges contain a 10 mg bed of Ludger Electron Interaction Resin (EIR). This unique material contains very flat regions of pi-orbital electrons that interact with hydrophilic organic compounds including glycans.

The resin can capture a wide range of glycans from complex mixtures while salts and certain detergents pass through. The glycans are then eluted from the cartridge using a simple solvent system. The cartridge housing is constructed of polypropylene and is compatible with many automated sample preparation systems.


Glycan Standards

Free Glycans A4 Family Glycans -NA4 Glycan -NGA4 Glycan

A3 Family Glycans -A3 Glycan -NA3 Glycan

-NGA3 Glycan

A2F Family Glycans -A2F Glycan-A1F Glycan -NA2F Glycan

NGA2F Glycan

A2 Family Glycans -A2 Glycan -A1 Glycan -NGA2 Glycan -M3N2 Glycan

Miscellaneous N-Linked Glycans -Hybrid Glycans

Oligomannose Family Glycans -Man-5 Glycan -Man-6 Glycan -Man-7 Glycan -Man-8 Glycan -Man-9 Glycan

N – Glycans are used as standards for glycoanalysis or as biological reagents. Glycans have been implicated in a variety of disease states. N-glycans are used as standards for glycoanalysis.

A wide variety of N-glycans are available.


Fluorescent Labeling of Glycans

Free Glycans

Labeled Glycans

The free glycans are tagged with fluorescent and UV active dye labels to assay them.

Glycan labeling kits –Glycan labeling kits –

Ludger Tag 2-AA Kit

Ludger Tag 2-AB Kit

Ludger Tag AA-AC Kit

Ludger Tag 2-AP


Post Labeling Clean Up

Labeled Glycans

Post labeling the glycans need to be cleaned up for excess labeling reagents following reductive amination using the LudgerTag glycan labeling system.

LudgerClean S Glycan Purification Cartridges are used to cleanup the labeled glycans.

LudgerClean S Glycan Purification Cartridges contain a hydrophilic glycan absorption disc that binds labeled glycans allowing removal of free dye and other non-carbohydrate material.

The cartridge housing is constructed of polypropylene and is compatible with many automated sample preparation systems.


Assaying Labeled Glycans

Labeled Glycans

Labeled glycans are assayed by HPLC, mass spectrophotometry, LC-MS, carbohydrate gel electrophoresis.

HPLC columns are available from Ludger for HPLC analysis.

The LudgerSep HPLC columns are designed for analysis and purification of a range of glycans including fluorophore tagged glycans prepared using LudgerSep glycan labeling kits and unlabeled glycans.

LudgerSep N1 Amide HPLC Column contain particles coated in a robust amide polymer. These bind to glycans under conditions of high organic solvent. Glycans are separated and eluted from the column using solvent gradients with decreasing organic solvent content.

Applications for Ludger HPLC column include the analysis and purification of glycans from complex mixtures, rapid profiling or fingerprinting of glycan populations, monosacharide composition analysis, and LC-MS.


Labeled Glycans

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Glossary

Carbohydrates: The largest class of organic compounds, including starches, glycogens, cellulose, gums, and simple sugars. Carbohydrates are composed of carbon, hydrogen, and oxygen in a ratio of Cn(H2O)n. [MeSH]

It has been estimated that about 0.5-1.0% of the translated mammalian genome participates in oligosaccharide production and function (Varki and Marth 1995). The magnitude of this genomic commitment to glycoconjugate biosynthesis is similar to that of protein phosphorylation, and may have as broad an impact on metazoan biology as the latter. In eukaryotes, protein and lipid glycosylation changes are observed during development, differentiation and importantly, numerous disease states. These differences are even being used as specific markers for diseases. Infectious diseases, and immune response are deeply imbued and dependent on carbohydrate residues.[Center for Structural Biology, Univ. of New Hampshire, US, Oct. 2000]

Glycoproteins: Glycoproteins are complexes in which carbohydrates are attached covalently to asparagine (N-glycans) or serine/ threonine (O-lycans) residues of peptides. Polysaccharides: Compounds consisting of a large number of monosaccharides linked glycosidically. This term is commonly used only for those containing more than ten monosaccharide residues. Also called glycans. Endoglycosidase An enzyme that catalyzes the cleavage of an internal glycosidic linkage in an oligosaccharide or polysaccharide.

Exoglycosidase An enzyme that cleaves a monosaccharide from the outer (nonreducing) end of an oligosaccharide, polysaccharide, or glycoconjugate. Glycan A generic term for any sugar or assembly of sugars, in free form or attached to another molecule, used interchangeably in this book with saccharide or carbohydrate.

Glycosylation The enzyme-catalyzed covalent attachment of a carbohydrate to a polypeptide, lipid, polynucleotide, carbohydrate, or other organic compound, generally catalyzed by glycosyltransferases, utilizing specific sugar nucleotide donor substrates.

N-glycan (N-linked Oligosaccharide, N-linked Glycan) Glycan covalently linked to an asparagine residue of a polypeptide chain in the consensus sequence: -Asn-X-Ser/Thr.


Glossary

Oligosaccharide Linear or branched chain of monosaccharides attached to one another via glycosidic linkages. The number of monosaccharide units can vary; the term polysaccharide is usually reserved for large glycans with repeating units.

O-glycan (O-linked Oligosaccharide, O-linked Glycan) A glycan glycosidically linked to the hydroxyl group of the amino acids serine, threonine, tyrosine, or hydroxylysine.

Hydrazinolysis A chemical method that uses hydrazine to cleave amide bonds, e.g., the glycosylamine linkage between a sugar residue and asparagine or the acetamide bond in N-acetylhexosamines.


References

• Bayer, E.A., F. De Meester, T. Kulik and M. Wilchek.’Preparation of deglycosylated egg white avidin’. Appl. Biochem Biotech 53: 1-9 (1995)• Bigge, J.C.; Patel, T.P; Bruce, J.A.; Goulding, P.N.; Charles, S.M; Parekh, R.B. (1995) ‘Non-selective and efficient fluorescent labeling of glycans using 2- aminobenzamide and anthranilic acid’. Analytical Biochemistry 230: 229-238• Charlwood, J.; Birrell, H. Gribble, A.; Burdes, V.; Tolson, D.; Camilleri, P. (2000) ‘A probe for the versatile analysis and characterization of N-linked oligosaccha rides’Analytical Chemistry 72: 1453-1461• Fan, J.Q.; Huynh, L.H.; Lee, Y.C. (1995). ‘Purification of 2-aminopyridine derivatives of oligosaccharides and related compounds by cation-exchange chromatography. Analytical Biochemistry 232:65-68• Guile, G.R.; Rudd, P.M.; Wing, D.R.; Prime, S.B.; Dwek, R.A. (1996) ‘A rapid and high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles’. Analytical Biochemistry 240: 210-226• Hardy, M.R. (1997) ‘Glycan labeling with the fluorophores 2-aminobenzamide and anthranilic acid’ in ‘Techniques in Glycobiology’, edited by Townsend, R.R and Hotchkiss, A.T.. Marcel Dekker Inc, New York• Iwase, H.; Ishii-Karakasa, I.; Urata, T.; Saito, T.; Saito, T.; Ho.a, K. (1990) ‘Extraction method for preparing pyridylamino sugar derivates and application to porcine gastric mucus glycoprotein analysis’. Analytical Biochemistry 188: 200-202• Tarentino, A .L. , C.M. Gomez an d T.H . Plummer, Jr.’Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F’. Biochemistry 24: 4665-4671 (1985)• Townsend, R.R.; Lipniunas, P.H.; Bigge, C.; Ventom, A.; Parekh, R. (1996) ‘Multimode high-performance liquid chromatography of fluorescently labeled oligosaccharides from glycoproteins’. Analytical Biochemistry 239: 200-207



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