Description the kit contains reagents for the conjugation of dye to the free reducing end of the glycan by a reductive amination reaction. The glycanbuilder will constitute the main interface for structure searches and the display of the results in the eurocarbdb databases. Read this article published by analytical chemistry where a team of scientists from amgen explore a quantitative highthroughput msbased mapping approach using stable isotope 2aminobenzoic acid 2 aa. Glycomics is the comprehensive study of glycomes the entire complement of sugars, whether free or present in more complex molecules of an organism, including genetic, physiologic, pathologic, and. Home glycan analysis and quantification glycan profiling and identification we offer uplcfldms of 2ab, 2aa or procainamide labeled glycans profiling service of cell, tissue or glycoprotein samples, and malditof ms based glycan structure identification, including. Identification and quantification of protein glycosylation. In addition, we offer complete kits for both 2 ab and rapifluorms labeling in a variety of sample sizes for low throughput to high throughput. However, it also available commercially as stable isotopic 12 c62aminobenzoic acid 12 c62 aa and c62 aa. Apart from the internal crossvalidation, classification efficiency was evaluated for the ms data using clinpro tools software. Exports images of glycan structures, fragments, and spectra as jpeg or png files. Glycoprofile 2aa labeling kit has been used in the labeling of fluorescently derivatized nglycans from chinese hamster ovary cho cells. In addition, we offer complete kits for both 2ab and rapifluor. O glycan release can be performed with hydrazinolysis or beta elimination, and a recent study has made use of household bleach for this purpose 33. Dried sample nglycans and c 2 aa labeled glycan standard were dissolved in h2o and 2 aa labeling solution 100 mgml picoline borane, 50 mgml 2 aa in a 7.
Purchase of this 2 ab labeling kit grants a use sublicense under glykos 2 ab patents. Ludgertag 2 aa glycan labeling kits ltkaaa2, sodium cyanoborohydride reductant. Sample cleanup devices with hilic aminopropyle sorbent. Of the fluorescent labels available, 2 aminobenzamide 2 ab and 2 aminobenzoic acid 2 aa are most widely used for oligosaccharide profiling. Accordingly, several kits are available for nglycan labeling. Glycomics is the systematic study of all glycan structures of a given cell type or organism and is a subset of glycobiology. The labeling chemistry for quaternary amine containing isobaric tag reagents according to the disclosure may be conducted in an analogous manner as 2 aa 2ab 2aminobenzoic acid 2 aa or 2aminobenzamide 2ab, so wellestablished protocols for 2 aa. Assessment, development, and deployment of workflows from sample prep through final reports. Improved fluorescent labeling efficiency of nlinked, high. Enzymatic digestion and mass spectroscopies of nlinked. Enzymatic release of the nglycans from glycoproteins. You can now compare isotopically distinct forms of the same. The labeling occurs at the reducing end of the oligosaccharide, which ensures each glycan will react with only one 2 ab molecule, making this approach also suitable for quantitative analysis. Automated identification and quantitation of stable.
Using 8aminopyrene1,3,6trisulfonic acid apts for analysis of glycoproteins. Similar to the 2ab label used in glycan characterization, the 2aminobenzoic acid 2 aa label offers many of the same benefits for lcms analysis. Lc coupled to the fluorescence detector has become standard technique for characterization of 2 aa and 2ab labeled nglycans. Application for labeling of free glycans with 2 aminobenzoic acid 2 aa. The glycoworks kit offers a protocol for complete sample processing of released nglycans with either 2ab or 2 aa labeling.
Advancements in mass spectrometrybased glycoproteomics and glycomics haojie lu. This kit contains 2picoline borane as the reductant. Application for labeling of free glycans with 2aminobenzoic acid 2aa. Lc coupled to the fluorescence detector has become standard technique for characterization of 2 aa and 2 ab labeled nglycans. These standards come prelabeled with 2aa, ready to reconstitute and inject. The fluorescent molecule is conjugated to the glycan reducing end via reductive amination.
This core structure of nlinked glycans, thus, consists of 14 residues 3 glucose, 9 mannose, and 2 nacetylglucosamine. Home glycan analysis and quantification glycan profiling and identification we offer uplcfldms of 2ab, 2 aa or procainamide labeled glycans profiling service of cell, tissue or glycoprotein samples, and. One of the commonly used labeling methods employs reducing end derivatization and reductive amination with compounds, such as 2aminobenzamide 2ab and 2aminobenzoic acid 2 aa. The glycanbuilder has been developed for the eurocarbdb project with the purpose of creating a fast, intuitive and flexible software tool for building and displaying glycan structures. Fluorescence labeling and purification of 2 aa derivatized oligosaccharides. Labeled n glycan appslab methods featured nibrt application. Glycan release and labeling kit pn 760523 high resolution protein labchip pn 760524, labchip gxii, labchip gxii touch ht. Area under curve of extracted ion chromatograms of the identified glycans were used as their measure of quantity in the samples. Similar to the 2 ab label used in glycan characterization, the 2 aminobenzoic acid 2 aa label offers many of the same benefits for lcms analysis. The resulting mixture of glycans can then be chemoselectively derivatized with a fluorescent label8 like 2aminobenzoic acid 2 aa, 2aminobenzamide 2ab, or 9aminopyrene1,4,6trisulfonic acid apts. They are the most referenced glycan label for biopharmaceutical analysis. A mass shift of only 4 da was achieved, which may result in a partial overlap of glycan pair isotope envelopes in massspectrometric. The fluorescent molecule is conjugated to the glycan reducing end via reductive amination 42. Glycan labeling strategies and their use in identification and quantification.
Labeled glycans may be analyzed by highsensitivity fluorescence detection or monitoring of uvabsorbance during various chromatographic and structure sequence analyses, as well as mass spectrometry. Preparation of released nglycan samples from monoclonal. Fast glycan labeling and analysis kit application guide. Two formats offered, one for highthroughput which is a 96well plate and the other for low volume users in a 1 cc cartridge format. We offer standards in a wide variety of labels, 2ab, 2 aa and rapifluorms. Ms2 spectrum of the 2aalabeled g3f nglycan from mab3 openi. Stableisotope labeling the software can identify glycans using msms data of samples treated with stable isotopic labeling techniques. This not only consumes a large amount of the users. Waters glycan standards and kits are for released nglycan labeling. Development of a rapid and robust nglycan profiling. Identification and quantitation of 2aa derivatized nglycans from infliximab using uhplc coupled to a thermo scientific q exactive plus mass spectrometer with simglycan software. Ludgertag 2aa 2aminobenzoic acid glycan labeling kit.
The most commonly used fluorophores are 2aminobenzoic acid 2 aa. The hydrophilicity of a 2 ab glycan increases with the molecular weight, hence with the number of carbohydrate monomers. Glycan biomarkers for alzheimer disease correlate with t. Procainamide labeled glycans are commonly used in hilicuhplc and esims assays. Software to manage lab inventories and assets, find location, determine ownership, and more. A 50mgml solution of 2ab or 2 aa labeling reagent was prepared by dissolution of the solid reagent in a dmsohoac. Waters glycan standards and kits are for released n glycan labeling. Mar 12, 2010 in an alternative attempt, oligosaccharides from chondroitin sulfates were labeled with h 4 2 aa or d 4 2 aa 41, 42, and separation by size exclusion was performed to avoid possible shifts in retention time. Accordingly, several kits are available for n glycan labeling. Fast glycan labeling and analysis kit for the ma m plus pharmaceutical analysis system application guide.
For labeling of free glycans with 2 aminobenzoic acid 2 aa. Ab labeling kits contain reagents for labeling glycans at their reducing ends by reductive amination. Frontiers glycan microarrays as chemical tools for. Glycan arrays, like that offered by the consortium for functional glycomics and z biotech llc, contain carbohydrate compounds that can be screened with lectins or antibodies to define. The dmb sialic acid labeling kit has been developed for the qualitative analysis of sialic acids. Jan 31, 20 this selectivity of the reversedphase chromatography in combination with 2 aa labeling enables separation of a variety of structural isomers of different types of n glycan and, more important, separates the nglycans into seven different groups. Magnetic beadmediated capture of the released glycans. It should be noted that another approach using 2aminobenzamide 2ab and 2aminobenzoic acid 2 aa has also been developed, whereby glycans reductively labeled with 2ab can be isolated by chromatography and then directly covalently coupled to epoxyactivated glass slides via secondary amine chemistry to generate 2ab or 2 aa. Of the fluorescent labels available, 2aminobenzamide 2ab and 2aminobenzoic acid 2 aa are most widely used for oligosaccharide profiling. Mass spectrometricbased stable isotopic 2aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics article in analytical chemistry 824. Higgins glycosolutions corporation, worcester, ma results. As mentioned above, several reductive amination reagents have been effectively employed for the quantification of glycans, such as pa, 2 aa, 2ab, and others. Services including instrument software qualifications, consulting and custom validations focused on data integrity.
You can now compare isotopically distinct forms of the same glycan structure originating from different biological samples such as control and diseased. Glyko 2aa conserved trimannosyl core substituted with fucose2aa m3n2f nglycan labeled with 2aminobenzoic acid. Sign up for our newsletter subscribe to our newsletter and always be the first to hear about what is happening. Comprehensive nglycan profiling of cetuximab biosimilar. Ms2 spectrum of the 2 aa labeled g3f n glycan from mab3. The glyko 2aa human igg nlinked glycan library consists of complex biantennary oligosaccharides consistent. View protocol picture guide as a pdf view product documentation as pdf view certificate of analysis as pdf. Improved fluorescent labeling efficiency of nlinked, highmannose oligosaccharides. The glycoworks kit offers a protocol for complete sample processing of released nglycans with either 2ab or 2aa labeling. Sign up for our newsletter subscribe to our newsletter and always be the first to. In chromatography, fluorescent labeling of the glycan moiety usually improves the detection. Pdf comparison of orthogonal chromatographic and lectin.
Glycomics is the comprehensive study of glycomes the entire complement of sugars, whether free or present in more complex molecules of an organism, including genetic, physiologic, pathologic, and other aspects. Ludgertag procainamide glycan labeling kits click for a presentation and more information. Integrated lcms workflow for the analysis of labeled and. Glyx nglycan rapid release and labeling with 2ab express kit. Dye properties mass 7 fluorescence, ex free dye 320 nm, ex glycandye conjugate 360 nm em 420 nm. Moreover the flexibility of the workflow allows analysis on both ce and lc with similar profile and comparable quality. Haojie lu, ying zhang, pengyuan yang, advancements in mass spectrometrybased glycoproteomics. Procainamide gives 3 times more response in hplc assays compared to 2ab labelled glycans and 30 times more response for esims. Infliximab, a monoclonal igg1 antibody, is designed to target inflammatory cytokines mainly tumor necrosis factoralpha.
However, it also available commercially as stable isotopic 12 c6 2 aminobenzoic acid 12 c6 2 aa and c6 2 aa variants and thus can be used for relative quantitation experiments. Uhplc analysis of 2ablabeled dextran ladder and assignment. New ludgertag 2ab and 2aa glycan labeling kits with non. Advancements in mass spectrometrybased glycoproteomics. Creates reports that include glycan characteristics such as id, sequence, name, composition, mass, precursor ion mz, class, reaction, pathway, glycan image and theoretical fragments with corresponding mass and mz values. The fast glycan labeling and analysis kit is for laboratory use onlyk introduction the protocol consists of the following steps. Sbio offers a line of cell culture labware with demonstrated high performance for glycan analysis. Get instant access to over a dozen ic and uhplc methods for 2 aa, 2ab labeled nglycans from therapeutic proteins.
Reversedphase liquidchromatographic mass spectrometric n. Importantly, 2 aa labeling of glycans can be performed under aqueous conditions, which allows labeling after glycan release without prior sample workup 94. It is proposed that the orthocarboxylic acid functionality of 2 aa. The following suggestions and data are based on information we believe to be reliable. B nglycans of the glycopeptides after each preparation step ad were analyzed in a linear negative ion mode after 2 aa labeling which adds 121 da by using malditof mass. Sign up for our newsletter subscribe to our newsletter and always be the first to hear. Add 20 l of 2ab labeling reagent to 50 g of free glycans and vortex to mix the solution.
Dried sample nglycans and c 2aa labeled glycan standard were dissolved in h2o and 2aa labeling solution 100 mgml picoline borane, 50 mgml 2aa in a 7. Additionally similarity of nglycan profiles from apts labeling to that of 2 aa. The labeling occurs at the reducing end of the oligosaccharide, which ensures each glycan will react with only one 2ab molecule, making this approach also suitable for quantitative analysis. Labels with both higher fluorescence and ms sensitivity have been introduced recently4. Existing 2 aa and 2 ab labeling kits use sodium cyanoborohydride as a reducing agent during glycan labeling. Labeling is performed at the reducing end of the glycan by reductive amination, with the most common labels being 2aminobenzamide 2ab or 2aminobenzoic acid 2 aa. Shop sbio products sbio glycan analysis services and. The glycoworks kit offers a protocol for complete sample processing of released nglycans with either 2 ab or 2 aa labeling. The glycan accounts for glycan is transferred en bloc by the glycosyltransferase oligosaccharyltransferase to a nascent peptide chain, within the reticular lumen.
A new high throughput labeling kit for the labeling of free glycans with 2aminobenzamide acid 2ab. We offer standards in a wide variety of labels, 2 ab, 2 aa and rapifluorms. Disadvantage of fluorescence detection compared to ms detection is that glycans have to be labeled before being detected. Get instant access to over a dozen ic and uhplc methods for 2 aa, 2 ab labeled nglycans from therapeutic proteins. Mass spectrometricbased stable isotopic 2aminobenzoic. Or access our latest featured application from nibrt ireland on fast profiling of. Glycan labeling strategies and their use in identification. Fluorescence labeling and purification of 2aa derivatized oligosaccharides. Glycan analysis and quantification asparia glycomics. The term glycomics is derived from the chemical prefix. Comparison of orthogonal chromatographic and lectinaffinity microarray methods for glycan profiling of a therapeutic monoclonal antibody. Oh glycan reductant oh h n ho o ho nhac n pp0520 nhac ro oh h. By accepting delivery of the glycoprofile 2 ab labeling kit materials and by subsequently using it in glycan analysis, recipient agrees to be bound by the following terms and restrictions.
Glycan analysis information thermo fisher scientific in. Nglycans of complex glycosylated biopharmaceuticals and. Glycan labeling strategies and their use in identification and quantification 3467. Oligosaccharide profiling of olinked oligosaccharides labeled with 2 aminobenzoic acid 2 aa elisabeth a.