Processes ms n data to resolve glycan heterogeneity, branching patterns, and isobaric structures. The company conducts research and development in new glycotechnology and produces a range of glycoprofiling tools for the biotechnology and pharmaceuticals industries. The applied biosystems glycanassure hyperformance apts system is a rapid nlinked glycan release, labeling, and clean up system that provides flexibility in sample preparation and in glycan analysis while ensuring accurate and consistent data through complete deglycosylation and preservation of sialic acid. One mass spectrometer for glycan and intact glycoform characterization. Magnetic beadmediated capture of the released glycans. A new high throughput labeling kit for the labeling of free glycans with 2aminobenzamide acid 2ab. Polymer beads are densely covered with hydrazide groups to facilitate glycan capture.
The 2ablabeled glycans were cleaned up using ludgerclean s cartridges ludger. 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. The five main glycans are identified via microchipce. The released glycans were incubated with labeling reagents for 3 h at 65 c. Labels various labels have been used for the reductive amination of glycans. Ludger ludgertagtm proc procainamide glycan labeling kit containing 2picoline borane each kit contains two sets of three vials ltaceticdmso01, ltproc01, ltpb01 take 150. Ludger specialises in analytical technology for biopharmaceutical glycosylation. Glycan labeling strategies and their use in identification.
Rapid analysis of nglycans on thelabchip gxii touch. The fast glycan labeling and analysis kit is for laboratory use onlyk introduction the protocol consists of the following steps. Waters and nibrt are codeveloping a new scientific library for rapifluorms labeled n glycans that identifies glycans. The study of saliva oglycosylation is receiving increasing attention due to. Glycosylation is the most common type of posttranslational protein modification khoury et al. The applied biosystems glycanassure hyperformance apts system is a rapid nlinked glycan release, labeling, and clean up system that provides flexibility in sample preparation and in glycan analysis. Glycotechnology and glycoprofiling tools ludgers products. Orela olinked glycan release kit qabio labeling kits. Robust automated highthroughput nglycan analysis using. Hplcbased analysis of serum nglycans on a 96well plate. Analysis of three epoetin alpha products by lc and lcms. Furthermore, comprehensive support for resolving glycopeptides using lcmsms glycopeptide data facilitates glycosylation studies. Glycosylation is a common posttranslational modification that affects the protein function through the attachment of glycans. The 2ab labeling solution was composed of acetic aciddmso2absodium cyanoborohydride 30.
The company conducts research and development in new glycotechnology and produces a range of glycoprofiling tools for the. This includes kits for release and labelling of monosaccharides or sialic acids, n or o glycan labelling kits and glycan clean up systems. We also offer system suitability standards and qualitative and quantitative. Simglycan software a product of premier biosoft predicts glycan and glycopeptide structures from sciex mass spectrometeracquired msms data, facilitating the identification of glycosylation sites and posttranslational modifications. Mix 20ul of 25sample and cy3 monoreactive dye 100ug labeling and incubate it for 1 hour at r. Fast glycan labeling and analysis kit application guide. Glyx n glycan rapid release and labeling with 2ab express kit utilizes a novel insolution enzymatic protein deglycosylation followed by express labeling of released n glycans with 2ab dye. Unlike dna replication and protein transcription, glycosylation is a nontemplatedriven process, 5,14 where the sugar residues form a multitude of arrangements. Nglycan release, labelling and cleanup were obtained from ludger.
The table below summarizes the applications, type of label, reductant method and analytical platform for each ludgertag kit. We also sell labelledunlabelled glycans, glycan hplc columns, endoglycosidases e. The glycanbuilder will constitute the main interface for structure searches and the display of the results in the eurocarbdb databases. This is the typical set of methods that we would use to analyse n glycans. For example, n glycan biosynthesis is shown both as a pathway map and a structure map in map00510. Ludger glycan analysis services more information could be find on ludger company website.
Glytoucan is the international glycan structure repository. Existing 2aa and 2ab labeling kits use sodium cyanoborohydride as a reducing agent during glycan labeling. Ludger t1 cartridges are designed for faster purification of glycans after labeling with the ludgertag 2ab and 2aa kits. Glycan labeling strategies and their use in identification and. Tm 2ab 2aminobenzamide glycan labeling kit containing. Fast glycan labeling and analysis kit for the ma m application guide fast glycan labeling. Labeled glycans may be followed by either highsensitivity fluorescence.
Labeling is performed at the reducing end of the glycan by reductive amination, with the most common labels. Procainamide labelled glycan standards ludgers products. Proc procainamide glycan labeling kit containing 2picoline borane ludger product code. Identification and separation of nglycans by hilicms. Up to seven levels of ms fragmentation can be needed to differentiate between glycan. Monoclonal antibody nglycan identification using msms library. Glycan analysis products and services welcome to ludger. Glycosylation is a posttranslational modification and, unlike transcription, is a nontemplatedriven enzymatic modification process its structural status depending largely on. The program is available in the form of web link below and standalone gui applications. Procainamide ludgertag labeling technique to permit glycan analysis by uhplc, esims, or lcesims. As a result, it is harder to explore o glycans using above software directly. This document provides the details of one standard set of methods used at ludger for glycan release and profiling of 2ab labelled n glycans.
Ludger ludgertag proc procainamide glycan labeling kit containing 2picoline borane each kit contains two sets of three vials ltaceticdmso01, ltproc01, ltpb01 take 150. Alterations of protein glycosylation are indicative of diseases and may occur through changes of the glycans alterations in monosaccharide composition, glycan structure or linkage, aberrant glycosylation and dynamics of microheterogeneities. Nglycan analysis of monoclonal antibodies and other. Ab labeling kits contain reagents for labeling glycans at their reducing ends by reductive amination.
Glycan analysis protein glycosylation can play an important role in defining in vivo bioactivity, stability, solubility and metabolic fate of biotherapeutics. Blotglyco is a sample preparation kit for glycan analysis of biological samples. One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans. Services including instrument software qualifications, consulting and custom validations focused on data integrity. Download simglycan a comprehensive and reliable software solution that can predict the structure of a glycan from the msms data aquired by mass spectrometry. Software that may be described in this document is furnished under a license agreement. Hplcbased analysis of serum nglycans on a 96well plate platform with dedicated database software article in analytical biochemistry 3761. Labeling method 1 purify the glycans if necessary, remove noncarbohydrate contaminants from the samples ludger product lceb10a6 2 transfer sample to reaction vial the kit is designed to label up to 25 nmols of glycans per reaction.
Characterization of 2ab labeled released nlinked glycans. Procainamide ludgertag labeling technique to permit glycan analysis by uhplc, esims, or lcesi. Agilent glyx n glycan rapid release and labeling with apts express kit 7 note glyx apts express heat block gx500 is requ ired for apts express labeling on the cleanup plate. Some of them contain an additional representation of glycan biosynthesis or degradation, called the glycan structure map. Following on from research performed by ludger ltd. Ludgers procainamide labeling system is suitable for nglycans, oglycans, gslglycans, heparin or any sugar with a reducing terminus and uses the same reductive amination labeling method that has been. Glycotechnology and glycoprofiling tools ludgers policies. Build mab n glycan accurate mass database and msms library glycan validation using msms library search different msms fragmentation patterns of g1f isomers 2. Our catalogue includes kits for glycan release, labelling and cleanup. Released oglycans were labeled with 2ab according to bigge and coworkers using a ludger 2ab glycan labeling kit. N glycans was then vacuum dried, and the dried glycan samples were labeled with the fluorophore 2ab.
Labeling is performed at the reducing end of the glycan by reductive amination, with the most common labels being 2aminobenzamide 2ab or 2aminobenzoic acid 2aa. It also supports the depiction of glycan and glycopeptide fragmentation. Comparison of procainamide and 2aminobenzamide labeling. Digestion pngase f carbohydrate labeling analysis on labchip gxii touch microchipce n glycan. Nglycan profiling analysis of a monoclonal antibody using. The lack of a comprehensive glycan database is a drawback since novel and rare structures are not identified using database search techniques. A new glycan fluorescent label, rapifluormstm,isusedto label nlinked glycans. Ludgertag procainamide glycan labeling kits click for a presentation and more information ltkprocvp24, 2pb reductant. Unions seventh framework program fp7healthf52011 under. Our vtag glycan kit enables release of nglycans from glycoproteins, nglycan labeling and clean up within 1 hour for a single igg type sample and 2 hours for 30 igg type samples timing for release by pngase f might need to be adjusted for complex type samples. Ludgertag glycan labeling kits are designed for the fluorophore or chromophore labeling of glycans with a free reducing terminus. Ludger tag 2aa monosaccharide release and labeling kit. Ludgertag procainamide glycan labeling kits click for a presentation and more information.
Released o glycans were labeled with 2ab according to bigge and coworkers using a ludger 2ab glycan labeling kit. Exports images of glycan structures, fragments, and spectra as jpeg or png files. Features include i sample immobilization in 96well plates, glycan release, and fluorescent labeling. Tm proc procainamide glycan labeling kit containing 2.
Glycan labeling ludgertag glycan labeling kit reagents are purified to analytical grade and are dispensed and sealed under clean, inert atmospheres. A hamilton microlab starlet liquidhandling robot was used for sample preparation of the nglycans in a similar manner to that previously published. The thermo scientific q exactive biopharma platform of orbitrapbased mass spectrometers add superior denatured and native ms intact analysis and subunit topmiddledown analysis capabilities to the most powerful benchtop peptide mapping instruments available when combined with thermo scientific biopharma finder software. An software package for glycan annotation and quantitation using lcesims conference paper pdf available october 2014 with 208 reads how we measure reads. Ludger nglycan profiling and indentification with vtag. Assessment, development, and deployment of workflows from sample prep through final reports. Strategy for glycan release, fluorescent labeling, nphplc profiling, structure assignments, and relative quantitation. Ludger standard n glycan release, 2ab labelling, cleanup and hilicuhplc methods. This repository is a freely available, uncurated registry for glycan structures that assigns globally unique accession numbers to any glycan independent of the level of information provided by the experimental method used to identify the structure s. Due to protonation, the resulting mass shift in the ms is 120 da tables 1, 2, 3. Specification for glycan profiling analysis service. This kit contains 2picoline borane as the reductant. Supports all thermo scientific ion trap and orbitrapbased instruments performing glycan characterization. Fast glycan labeling and analysis kit for the ma m plus pharmaceutical analysis system application guide.
Glycanmass is a tool which allows to calculate the mass of an oligosaccharide structure mass values disclaimer note. The company conducts research and development in new glycotechnology and produces a range of glycoprofiling tools for. Ludger standard nglycan release, 2ab labelling, cleanup. New ludgertag 2ab and 2aa glycan labeling kits with non. The team of specialists at ludger can provide glycoprofiling services and technology to help you engineer, optimise, measure and control the glycosylation of your therapeutic glycoprotein product. Released glycans have free reducing terminii to allow fluorescent tagging by reductive amination. Ludger tag monosaccharide release and labeling kit specifications. Labeling cleanup prior to hplc analysis of fluorescently labeled oligosaccharides is imperative for highest quality glycoprofiling analysis by hplc. Mixed glycan samples should contain between 100 picomoles to 50 nanomoles of purified glycans. The 2ab labeling of the glycans results in the mass of the glycans. Highthroughput analysis and automation for glycomics studies. This was accompanied by selection of suitable tecan hardware and plastic consumable. An overview of the experimental strategy used for releasing, labelling and sequencing.
Labeled glycan workflow thermo fisher scientific in. Ludgers bioquant products allow you to quantitate glycans in your samples simply and reliably. Each glycoprofile labeling kit contains sufficient reagents for labeling up to 36 samples. O glycans have more diverse core structures, which mean the space of candidate of glycans is larger than that of n glycans. Labchip gxii touch microchipce glycan profiling workflow. A comprehensive range of products for you to use in your glycoprofiling workflow inhouse, whether you use lc, ms, lcms, or ce. Product guide for ludgertagtm 2ab 2aminobenzamide glycan labeling kit ludger product code. Ludger s bioquant products allow you to quantitate glycans in your samples simply and reliably. Nlinked glycans are attached in the endoplasmic reticulum to the nitrogen n in the side chain of asparagine in the sequon. Software tools for glycan profiling pubmed central pmc. After the labelling procedure, the samples were puri. Labeled glycans may be followed by either highsensitivity. This innovative label improves flr and ms signals for glycan characterization and profiling analysis. Application for release and labeling of neutral and amino monosaccharides from glycoprotein therapeutics and prereleased glycans.
May 01, 2019 in the kegg pathway database the section on. Orela kit has been developed for olinked deglycosylation of glycoproteins. We also realize that glycan profiling is a relatively new and complex science. Suppression of peeling during the release of oglycans by. Glycan profiling analysis service using lectin microarrays lectin arrays at glycotechnica, we firmly believe that the era of glycomics has just begun. Another labeling strategy for quantification lies in the use of isobaric tags. In this process, glycans are released from the therapeutic glycoprotein then tagged with a fluorescent dye such as 2ab 2aminobenzamide or 2aa 2aminobenzoic acid by reductive amination prior to chromatography. Furthermore, as glycans with various different structures may be present in minute amounts in glycoprotein hydrolysates, their detection by uv absorbance may not be. 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. Glycans do not contain fluorophores and thus must be labeled with fluorescent tags prior to analysis. Tm product guide for ludgertag 2ab 2aminobenzamide glycan.
For labeling of free glycans with procainamide proc 2 picoline borane. Glycans are assembled by various glycosyltransferases along a pathway starting at the endoplasmic reticulum continuing to the golgi apparatus, and these structures are expressed on the membrane, or as secreted extracellular matrix elements or soluble glycoproteins lairson et al. We have developed database matching software autogu which. Therefore it is our strategy to work closely with each and every customer to help comprehend the data being generated. Oct 11, 2019 drawglycansnfg is a simple and robust drawing tool for displaying glycans and glycopeptides from iupaccondensed format text inputs. You can use glycomod to predict the possible oligosaccharide structures that occur on proteins from their experimentally determined masses. Figure 3 shows the 2ab labeling by reductive amination schiffs base intermediate not shown. Current software for automation of analysis mainly focuses on. After labeling the rfms glycans were subjected to uplcflr qtof ms analysis. Highthroughput glycan analysis of serum from ra patients a correlation between the amount of nongalactosylated g0 biantennary glycans on igg and ra has been well established since 1985 10. It is against the law to copyi modifyi or distribute the software on any i except as specifically allowed in the license. Enzymatic release of the nglycans from glycoproteins. The sequon is an asnxser or asnxthr sequence, where x is any amino acid except proline and the glycan may be composed of nacetylgalactosamine, galactose, neuraminic acid, nacetylglucosamine, fucose, mannose, and other monosaccharides.