The biology of carbohydrates, or glycobiology, is attracting growing attention in high impact research areas (neurobiology, development, immunology), and also in applied fields such as manufacturing of biotherapeutics and antibodies, and heparin analogs. Carbohydrates attached to glycolipids or glycoproteins determine the properties and fates of living cells, therefore the structural characterization of glycans and glycosylated proteins plays a crucial role in elucidating central biological processes.
Glycoproteins and other glycoconjugates are structurally diverse and virtually impossible to predict from genetic information alone. Analytical tools, including specific enzymes such as endoglycosidases and exoglycosidases, along with chromatography, mass spectrometry methods and specialized bioinformatics, allow us to define the composition of glycoconjugates in any given model system. However, descriptive analysis only gives a snapshot of the biological process of interest. In many cases, purifying a target from a recombinant expression system is the preferred method to understand the functional properties of a glycoprotein.
- What are Glycosidases and their uses?
- Which proteins are glycosylated?
- Can glycosidases be used in combination for extensive digestion?
- Can I analyze protein glycosylation with only benchtop instrumentation and reagents?
- Do I need to deglycosylate my protein sample for proteomic analysis?
- How can I separate a deglycosylated protein from the glycosidase?
- Is it possible to predict whether a protein is N- or O-glycosylated?
- What are the advantages of enzymatic deglycosylation?
- What are the best techniques to analyze N- and O-glycans?
- Glycan Analysis of Murine IgG2a by Enzymatic Digestion with PNGase F and Trypsin, Followed by Mass Spectrometric Analysis
- Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
- Remove iT PNGase F Effective Release and Recovery of Neutral and Sialylated N glycans
- Proteomics Fast and Efficient Antibody Deglycosylation using Rapid PNGase F
- Glycan Analysis of Murine IgG by Enzymatic Digestion with Endo S and PNGase F Followed by Mass Spectrometric Analysis
- Enzymatic Deglycosylation of a Protein Containing Core 1 O-glycans with O-Glycosidase
- Removal of terminal galactose from a glycoprotein containing tri and tetraantennary N linked sugars with 1 3 6 Galactosidase
The Structure, Function and Importance of Carbohydrates
Read about the structure, function, and importance of Carbohydrates from biology experts at NEB.
Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein
Glycomics: A rapidly evolving field with a sweet future
Glycans are widely distributed in nature and have physical, chemical, and biological properties that make them important players in areas such as biofuels, food, materials science, biotechnology, and pharmaceuticals.
N-Glycan Composition Profiling for Quality Testing of Biotherapeutics
O-glycan analysis of therapeutic proteins enabled by O-glycoprotease
This article from European Pharmaceutical Review describes tools that can be used for O-glycan analysis of therapeutic proteins.
- Glycoproteomics Brochure
- Glycoproteomics Technical Guide
- Rapid PNGase F Trifold
- Glycan Analyzer
- Endoglycosidase Selection Chart
- Detailed Characterization of Several Glycosidase Enzymes
- Glycobiology Unit Conversion Chart
- Improving Sample Workflow: Rapid PNGase F for Accurate Antibody Characterization (2015)
- Chuzel, L.O., Ganatra, M.B., Rapp, E., Henrissat, B., Taron, C.H. (2018) Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156) J Biol Chem; 293(47), 18138-18150. PubMedID: 30249617
- Vainauskas, S., Kirk, C.H., Petralia, L., Guthrie, E.P., McLeod, E., Bielik, A., Luebbers, A., Foster, J.M., Hokke, C.H., Rudd, P.M., Shi, X., Taron, C.H (2018) A novel broad specificity fucosidase capable of core a1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes Sci Rep; PubMedID: 29934601, DOI: 1038/s41598-018-27797-0
- O'Flaherty, Roisin, Harbison, Aoife M., Hanley, Philip J., Fadda, Elisa, Rudd, Pauline, Taron, Chris (2017) Aminoquinoline fluorescent labels obstruct efficient removal of N-glycan corex J Proteome Res; 16, 4237-4243.
- Albrecht, S., Vainauskas, S., Stöckmann, H., McManus, C., Taron, C.H. and Rudd, P.M. (2016) Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow. Anal Chem; May 3;88(9), 4795-802. Anal Chem.. PubMedID: 27033327
- Zhao H, Blazanovic K, Choi Y, Bailey-Kellogg C, Griswold KE (2014) Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris Appl Environ Microbiol; 80(9), 2746-53. PubMedID: 24561590, DOI: 10.1128/AEM.03914-13
- Möykkynen T, Coleman SK, Semenov A, Keinänen K (2014) The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization J Biol Chem; 289(19), 13197-205. PubMedID: 24652293, DOI: 10.1074/jbc.M113.526301
- Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S (2014) A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells PLoS One; 9(5), e96635. PubMedID: 24804975, DOI: 10.1371/journal.pone.0096635
- Kwon HM, Lee KH, Han BW, Han MR, Kim DH, Kim DE (2014) An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells PLoS One; 9(5), e97574. PubMedID: 24835440, DOI: 10.1371/journal.pone.0097574
- Rosenbaum EE, Vasiljevic E, Brehm KS, Colley NJ (2014) Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster PLoS Genet; 10(5), e1004349. PubMedID: 24785692, DOI: 10.1371/journal.pgen.1004349
- Rosenbaek LL, Kortenoeven ML, Aroankins TS, Fenton RA (2014) Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis J Biol Chem; 289(19), 13347-61. PubMedID: 24668812, DOI: 10.1074/jbc.M113.543710
- Wicht O, Burkard C, de Haan CA, van Kuppeveld FJ, Rottier PJ, Bosch BJ (2014) Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell J Virol; 88(9), 4943-52. PubMedID: 24554652, DOI: 10.1128/JVI.03451-13
- Haller G, Li P, Esch C, Hsu S, Goate AM, Steinbach JH (2014) Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior PLoS One; 9(5), e96753. PubMedID: 24804708, DOI: 10.1371/journal.pone.0096753
- Wright CR, Brown EL, Della-Gatta PA, Ward AC, Lynch GS, Russell AP (2014) G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle Front Physiol; 5, 170. PubMedID: 24822049, DOI: 10.3389/fphys.2014.00170
- Itahana Y, Han R, Barbier S, Lei Z, Rozen S, Itahana K (2014) The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense Oncogene; PubMedID: 24858040, DOI: 10.1038/onc.2014.119
- Arakel EC, Brandenburg S, Uchida K, Zhang H, Lin YW, Kohl T, Schrul B, Sulkin MS, Efimov IR, Nichols CG, Lehnart SE, Schwappach B (2014) Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes J Cell Sci; 127(Pt 9), 2106-19. PubMedID: 24569881, DOI: 10.1242/jcs.141440
- Botto L, Cunati D, Coco S, Sesana S, Bulbarelli A, Biasini E, Colombo L, Negro A, Chiesa R, Masserini M, Palestini P (2014) Role of lipid rafts and GM1 in the segregation and processing of prion protein PLoS One; 9(5), e98344. PubMedID: 24859148, DOI: 10.1371/journal.pone.0098344
- Velho, A.M., Jarvis, S.M. (2009) Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting Exp Cell Res; 315 , 2312-21 . PubMedID: 19379732
- Rasmussen, T.N., Plenge, P., Bay, T., Egebjerg, J., Gether, U. (2009) A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation Neuropharmacology; 57 , 287-94 . PubMedID: 19500602
- Gong, B., Cukan, M., Fisher, R., Li, H., Stadheim, T.A., Gerngross, T. (2009) Characterization of N-linked glycosylation on recombinant glycoproteins produced in Pichia pastoris using ESI-MS and MALDI-TOF Methods Mol Biol; 534 , 213-23 .
- Gefter, J.V., Shaufl, A.L., Fink, M.P., Delude, R.L. (2009) Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines Cell Tissue Res; 337 , 79-89 . PubMedID: 19415334
- Boeggeman, E., Ramakrishnan, B., Pasek, M., Manzoni, M., Puri, A., Loomis, K.H., Waybright, T.J., Qasba, P.K. (2009) Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection Bioconjugate Chem; 20 , 1228-36 . PubMedID: 19425533
- Wagner-Rousset, E., Bednarczyk, A., Bussat, M.C., Colas, O., Corvaïa, N., Schaeffer, C., Van Dorsselaer, A., Beck, A. (2008) The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies J Chromatogr B Analyt Technol Biomed Life Sci; 872, 23-37. PubMedID: 18672411
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Learn about glycobiology and its importance in clinical and diagnostic applications in this episode of NEB TV. Also, hear more about how NEB is setting the bar for product quality in this rapidly growing field.
Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans can be accomplished with the use of deglycosylation enzymes, which can provide complete sugar removal with no protein degradation.