Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis. Experience with lc analytical and characterisation techniques including charge heterogeneity, size exclusion and n linked oligosaccharide profiling. Hence, determining the nlinked glycosylation of an mab is an important part of its characterization, and the glycosylation of each lot of a mab biotherapeutic must be comparable to the glycosylation of the mab used in its clinical trials. Oligosaccharide analysis by highperformance anionexchange. For example, glycolipids have an important role in the immune.
Quantitative proteomics analysis reveals important roles. Combining rapifluorms and unifi software for a total n. Nlinked glycosylation, in which a glycan moiety is added to the amide. The pa 800 plus carbohydrate labeling and analysis assay contain reagents, buffers, and separation capillaries for the analysis of n linked oligosaccharides. Ultrasensitive profiling and sequencing of n linked oligosaccharides using standard dnasequencing equipment nico callewaert, steven geysens, francis molemans, and roland contreras1 unit of fundamental and applied molecular biology, department of molecular biology, ghent university and flanders interuniversity institute. We also analyzed oligosaccharides labeled with 3aa. Profiling nlinked oligosaccharides from igg by high. This technical note hpaepad nlinked oligosaccharide profiling. Microanalysis of n linked oligosaccharides in a glycoprotein by capillary liquid chromatographymass spectrometry and liquid chromatographytandem mass spectrometry. Comprehensive profiling of n linked glycosylation sites in hela cells using hydrazide enrichment helle malerod, robert l. In this study, we employed a quantitative proteomics analysis to unravel the overall regulatory mechanisms of nglycosylation at different developmental stages of m. Profiling the nlinked oligosaccharides of a glycoprotein is one of the. The gel used came preformed in the biorad nlinked profiling kit. Glycan profiling of n and olinked oligosaccharide profiling from animal, plant and bacteria.
Mass spectrometric profiling of nlinked oligosaccharides and. The structures on these sites are either biantennary or triantennary complex oligosaccharides. Dionex carbopac pa200 3 x 250 mm or dionex carbopac pa100 or dioenx carbopac pa1 4 x 250 mm. The gel was run in a biorad mini protean ii gel apparatus with the kits. Total nlinked oligosaccharide profiling method for recombinant monoclonal antibody rmab using capillary electrophoresis with laserinduced fluorescence detection celif and an approach for. Improved fluorescent labeling efficiency of nlinked, high. Here, we report a highly reproducible combination of a glycomic sample preparationsolid. The minimum neutral sugar content observed is six residues, consistent with molecules having oligosaccharide moieties on each heavy chain devoid of galactose and fucose, i. Carbohydrate analysis throughout the development of a protein. Transcriptome profiling of bovine milk oligosaccharide. We report an improved hpaepad method for igg oligosaccharide separation. Highperformance anionexchange chromatography with pulsed amperometric detection hpaepad is a wellestablished, powerful technique for glycan analysis. Key benefits to carbohydrate profiling using capillary electrophoresis include. Cell biology page 6 specifically attach m6p to a lysosome catalytic site will bind phosphorylated glcnac nacetylglucosamine to c6 of a mannose residue.
N glycans are typically released from glycoproteins by enzymes, while oglycans are released from glycoproteins by chemical methods. The gel used came preformed in the biorad n linked profiling kit. The analysis of glycosylation patterns on glycoprotein therapeutics such as. Endoglycosidase f 2 cleaves between the two n acetylglucosamine residues in the n linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one n acetylglucosamine residue remaining on the asparagine. Nlinked oligosaccharide profiling by hplc on porous graphitized carbon pgc. It involves the assembly of an oligosaccharide on a lipid carrier, dolichylpyrophosphate and the transfer of the oligosaccharide to selected asparagine residues of polypeptides that have entered the lumen of the er. Oligosaccharide profiling also referred to as mapping or fingerprinting is the best method for monitoring the consistency of the glycosylation of your molecule. Apr 01, 2001 ultrasensitive profiling and sequencing of n linked oligosaccharides using standard dnasequencing equipment nico callewaert unit of fundamental and applied molecular biology, department of molecular biology, ghent university and flanders interuniversity institute for biotechnology, k. Highph anionexchange chromatography with pulsed amperometric detection hpaecpad is a very powerful tool for the profiling of n linked oligosaccharides 1,2, only being limited by the. N linked glycosylation, is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom the amide nitrogen of an asparagine asn residue of a protein, in a process called n glycosylation, studied in biochemistry. Hence, determining the n linked glycosylation of an mab is an important part of its characterization, and the glycosylation of each lot of a mab biotherapeutic must be comparable to the glycosylation of the mab used in its clinical trials. Carbohydrate analysis throughout the development of a. With this method, oligosaccharides n linked andor o linked are released intact from the molecule and separated to create the profile.
With this method, oligosaccharides nlinked andor olinked are released intact from. Sep 15, 2017 a highperformance liquid chromatography based strategy for rapid, sensitive sequencing of n linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide. The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Electrophoretic methods for the analysis of nlinked. All eukaryotic cells express nlinked glycoproteins. These characteristics are required for labeling, separation and quantification of oligosaccharides released from glycoproteins.
Improved profiling of sialylated nlinked glycans by ion. Total n linked oligosaccharide profiling method for recombinant monoclonal antibody rmab using capillary electrophoresis with laserinduced fluorescence detection celif and an approach for. Order immediately from any provider with preestablished contracts. Nlinked oligosaccharide profiling by hplc on porous. Characterization of recombinant monoclonal antibody nlinked. Combining the power of accurate mass, reference glucose units, and unifi software for confident glycan assignments. Protein glycosylation of n linked glycans is actually a cotranslational event, occurring during protein synthesis. Ultrasensitive profiling and sequencing of nlinked oligosaccharides using standard dnasequencing equipment. Comprehensive profiling of nlinked glycosylation sites in. Glycosylation occurs most often when this consensus sequence occurs in a loop in the peptide. May 21, 2010 the study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. We used a motifx algorithm software to generate sequence logos 31. Glycans help to define activity, efficacy and immunogenicity of mabs.
The filter sorts compositions based upon the quantities of each monosaccharide present for a composition and classifies the oligosaccharide as neutral complex, anionic complex, and. The process of n linked glycosylation occurs cotranslationally, or concurrently while the proteins is being translated. Characterization of the n linked oligosaccharide glycan profile of a monoclonal antibody mab is an important component of consistent lot release for clinical studies and marketed product support. Endo f2 cleaves biantennary complex and to a lesser extent high mannose oligosaccharides.
Labelfree nglycan profiling ion chromatography for n linked and o linked glycan analysis high performance anionexchange chromatography with pulsed amperometric detection hpaepad on systems such as the dionex ics6000 hybrid hpic system, is a wellestablished analysis method to characterize released glycans. Improving characterization of monoclonal antibodies. This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylationrelated genes. Nlinked protein glycosylation in the endoplasmic reticulum er is a conserved two phase process in eukaryotic cells. Oligosaccharide profiling of olinked oligosaccharides. Comparative structural study of nlinked oligosaccharides of. Thus, oligosaccharide 3 having a gal residue at man. Unlabeled glycan workflow thermo fisher scientific us. Structures of nlinked ofigosaccharides the n or asparaginelinked oligosaccharides represent a diverse group of structures, that share a common biosynthetic pathway, and that contain a common core region oe man0tl,6man0tl,3 manb1,4glcnacb1,4glcnac. Nlinked glycan profiling of mature human milk by high. Sialic acids of a murine igg1s nlinked oligosaccharides were shown to. Nlinked glycosylation requires the consensus sequence asnxserthr. Profiling nlinked oligosaccharides from igg by highperformance anion exchange chromatography with pulsed amperometric detection.
Comparative structural study of nlinked oligosaccharides. Conformational analysis of asnlinked oligosaccharides. Oct 01, 2004 the oligosaccharide 3 having a gal residue at man. Detect released oligosaccharides using the eagle eye ii. Highph anionexchange chromatography with pulsed amperometric detection hpaecpad is a very powerful tool for the profiling of nlinked oligosaccharides 1, 2, only being limited by the concentrated sodium hydroxide and sodium acetate required to achieve the separation and sensitive detection. Comparative characterization of the fc domain nglycosylation.
Mar 10, 2015 profiling glycoprotein nlinked oligosaccharide by capillary electrophoresis. Highph anionexchange chromatography with pulsed amperometric detection hpaecpad is a very powerful tool for the profiling of n linked oligosaccharides 1, 2, only being limited by the concentrated sodium hydroxide and sodium acetate required to achieve the separation and sensitive detection. Profiling glycoprotein nlinked oligosaccharide by capillary electrophoresis. Structures of n linked ofigosaccharides the n or asparagine linked oligosaccharides represent a diverse group of structures, that share a common biosynthetic pathway, and that contain a common core region oe man0tl,6man0tl,3 manb1,4glcnacb1,4glcnac. N linked glycosylation involves oligosaccharide attachment to asparagine via a beta linkage to the amine nitrogen of the side chain. The gel contained 8aminonaphthalene1,3,6trisulphonate ants 4labeled, nlinked oligosaccharides, released from various glycoproteins, separated by oligosaccharide page. In glycan profiling we can release, purify and derivate of n and olinked glycans by enzymatic or chemical methods such as betaelimination and hydrazinolysis prior to ms.
It is important to identify and quantify both n and olinked glycans of glycoproteins to. Highperformance anionexchange chromatography with pulsed amperometric detection hpaepad is an effective tool for determining glycans present in glycoprotein therapeutics. The biochemical role of oligosaccharides in glycoproteins has been examined for intracellular trafficking, proteinase susceptibility, molecular conformation, protein stability and proteinprotein interactions. Pdf nlinked oligosaccharide profiling by hplc on porous. N linked protein glycosylation in the endoplasmic reticulum er is a conserved two phase process in eukaryotic cells. Role of nlinked oligosaccharide chains in the processing and antigenicity of measles virus haemagglutinin protein a. Ultrasensitive profiling and sequencing of nlinked oligosaccharides using standard dnasequencing equipment nico callewaert, steven geysens, francis molemans, and roland contreras1 unit of fundamental and applied molecular biology, department of molecular biology, ghent university and flanders interuniversity institute.
Role of nlinked oligosaccharide chains in the processing and. Kakehi, capillary electrophoresis with laserinduced fluorescence detection for detailed studies on nlinked oligosaccharide profile of therapeutic recombinant monoclonal antibodies, journal of chromatography a, vol. Fetuin is a wellcharacterized glycoprotein containing both nlinked oligosaccharides and olinked oligosaccharides. The role of nlinked oligosaccharides in glycoprotein function. The crystal structure of the singlesubunit oligosaccharyltransferase pglb in complex with acceptor peptide and a synthetic lipidlinked oligosaccharide analog reveals a key intermediate in the. David brazil data analyst privacy analytics linkedin. Good profiling of a recombinant glycoproteins asparaginelinked nlinked glycans requires highresolution separation and reliable identification of released glycans. Oct 23, 2017 the crystal structure of the singlesubunit oligosaccharyltransferase pglb in complex with acceptor peptide and a synthetic lipid linked oligosaccharide analog reveals a key intermediate in the. Processing of nlinked oligosaccharide chains on glycoproteins within the 3 golgi cisternae.
Nglycans are typically released from glycoproteins by enzymes, while oglycans are released from glycoproteins by chemical methods. Nlinked oligosaccharide profiling by hpaecpad springerlink. Characterization of the nlinked oligosaccharide glycan profile of a monoclonal antibody mab is an important component of consistent lot release for clinical studies and marketed product support. Automated nglycosylation sequencing of biopharmaceuticals. Vast array control software reductive amination preparative work pulse. Quantitative proteomics analysis reveals important roles of n. Oligosaccharides can have many functions including cell recognition and cell binding. Glycan profiling of n and olinked oligosaccharide profiling from animal,plant. Nlinked oligosaccharide profiling by hplc on porous graphitized. Nlinked glycosylation, in which a glycan moiety is added to the amide group of an asparagine residue, is an abundant modification known to be essential for m. N linked glycosylation requires the consensus sequence asnxserthr. The pa 800 plus carbohydrate labeling and analysis assay contain reagents, buffers, and separation capillaries for the analysis of nlinked oligosaccharides. Two common types of glycans, nlinked and olinked, have been extensively analyzed in the last decades. In glycan profiling we can release, purify and derivate of n and olinked glycans by enzymatic or chemical methods such as betaelimination and hydrazinolysis prior to ms profiling.
It is important to identify and quantify both n and o linked glycans of glycoproteins to. Ultrasensitive profiling and sequencing of nlinked. The gel contained 8aminonaphthalene1,3,6trisulphonate ants 4labeled, n linked oligosaccharides, released from various glycoproteins, separated by oligosaccharide page. For attachment to occur the amino acid motif usually needs to be asnx. Glycoworks rapifluorms nglycan kit 96 sample care and use manual. Comprehensive profiling of nlinked glycosylation sites in hela cells using hydrazide enrichment. Highph anionexchange chromatography with pulsed amperometric detection hpaecpad is a very powerful tool for the profiling of nlinked oligosaccharides 1,2, only being limited by the. Nlinked protein glycosylation in the er sciencedirect. Nlinked glycans are covalently attached to the protein at asparagine asn residues this most often occurs when the new protein is being translated and transported into the er. Automated nglycosylation sequencing of biopharmaceuticals by.
A study of the nlinked oligosaccharide structures of. Simultaneous analyses of nlinked and olinked glycans of. Quantification of glycomicprofile changes is becoming increasingly important in the search for disease biomarkers. Profiling nlinked oligosaccharides from igg by highperformance. Fresh milk samples n 32 were collected from four holstein and jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. Glycans play critical roles in a number of biological activities. Profiling analysis of oligosaccharides in antibody. Manage projects, exchange data, securely communicate. Molecular basis of lipidlinked oligosaccharide recognition. Glycan profiling of n and olinked oligosaccharide analytical. C profiling of nlinked oligosaccharides released from polyclonal human igg and a mab sample.
Nov 04, 2009 oligosaccharide profiling also referred to as mapping or fingerprinting is the best method for monitoring the consistency of the glycosylation of your molecule. Endoglycosidase f 2 cleaves between the two nacetylglucosamine residues in the nlinked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one nacetylglucosamine residue remaining on the asparagine. Experience with lc analytical and characterisation techniques including charge heterogeneity, size exclusion and nlinked oligosaccharide profiling. Method denaturation of protein with sdsme solution at 70c pngase digestionremoves n. Oligosaccharides are separated on the basis of charge i. Characterization of recombinant monoclonal antibody n. Experience with capillary electrophoresis analysis and a variety of compendial release techniques including osmolarity and turbidity. Oligosaccharide profiles ofmonoclonal iggofeach subclass the n acetylneuraminic sialic acid and minimum neutral sugar contents of sample proteins are given in table 1. Role of nlinked oligosaccharide chains in the processing. Profiling nlinked oligosaccharides from igg by highperformance anionexchange chromatography with pulsed amperometric detection. This technical note hpaepad n linked oligosaccharide profiling. Two common types of glycans, n linked and o linked, have been extensively analyzed in the last decades. Nlinked carbohydrate profiling of etanercept enbrel analyzed by using standard uplc upper trace and capillary gel electrophoresis lower trace separation parameters. Figure 3 compares the cgelif and uplcfl analysis of apts and 2ab labeled etanercept nglycans, respectively.
Microanalysis of nlinked oligosaccharides in a glycoprotein by capillary liquid chromatographymass spectrometry and liquid chromatographytandem mass spectrometry. Changes in oligosaccharide structures are associated with many physiological and pathological events. C profiling of nlinked oligosaccharides released from polyclonal. Role of n linked oligosaccharide chains in the processing and antigenicity of measles virus haemagglutinin protein a.
Nlinked oligosaccharides obtained from total serum of mice with. Mabs, in most instances, possess a conserved nlinked glycosylation site on each of the c h 2 domains of the fc portion of the heavy chain of the. Protein glycosylation of nlinked glycans is actually a cotranslational event, occurring during protein synthesis. Software for automated interpretation of mass spectrometry data.
181 1095 988 358 76 414 336 301 1212 298 361 531 353 1523 450 1150 680 826 1218 1000 233 702 819 60 495 808 72 1233 1365 284 159 1395 982 81 1316 1035 1068 41 1115 581 438 967 585