Method comparison for N-glycan profiling: Towards the standardization of glycoanalytical technologies for cell line analysis

Autoři: Maximilianos Kotsias aff001;  Athanasios Blanas aff002;  Sandra J. van Vliet aff002;  Martina Pirro aff003;  Daniel I. R. Spencer aff001;  Radoslaw P. Kozak aff001
Působiště autorů: Ludger Ltd., Culham Science Centre, Abingdon, Oxfordshire, England, United Kingdom aff001;  Amsterdam UMC, Vrije Universiteit Amsterdam, Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam, The Netherlands aff002;  Leiden University Medical Centre, Centre for Proteomics and Metabolomics, Leiden, The Netherlands aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.

Klíčová slova:

Blood plasma – Colorectal cancer – Consortia – Galactose – Glycosylation – HT29 cells – Hexoses – Mannose


1. Schwarz F, Aebi M. Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol [Internet]. Elsevier Ltd; 2011;21(5):576–82. Available from: doi: 10.1016/ 21978957

2. Singh S, Darbari H, Bhattacharjee K, Verma S. Open source NLG systems: A survey with a vision to design a true NLG system. Int J Control Theory Appl. 2016;9(10):4409–21.

3. Salimonu LS, Johnson AOK, Williams AIO, Iyabo Adeleye G, Osunkoya BO. Phagocyte function in protein-calorie malnutrition. Nutr Res. 1982;2(4):445–54.

4. Han BW, Kim WS, Lee JK, Lee SY, Park SH, Kim YI, et al. Angular dependency of magnetization losses in continuously transposed coated conductors for large current applications. Trans Korean Inst Electr Eng. 2010;59(1):51–6.

5. Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA. Glycosylation and the immune system. Science (80-). 2001;291(5512):2370–6.

6. Tanichi N, Korekane H. Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics. BMB Rep. 2011;44(12):772–81. doi: 10.5483/bmbrep.2011.44.12.772 22189679

7. Molinari M. N-glycan structure dictates extension of protein folding or onset of disposal. Nat Chem Biol. 2007;3(6):313–20. doi: 10.1038/nchembio880 17510649

8. Clerc F, Reiding KR, Jansen BC, Kammeijer GSM, Bondt A, Wuhrer M. Human plasma protein N-glycosylation. Glycoconj J. 2016;33(3):309–43. doi: 10.1007/s10719-015-9626-2 26555091

9. Lasebikan VO, Adebayo S. Need for Screening for Alcohol and Drugs in Emergency Trauma Units. East Afr Med J [Internet]. Elsevier B.V.; 2013;90(5):164–70. Available from: 26859007

10. Freeze HH. Genetic defects in the human glycome. Nat Rev Genet. 2006;7(7):537–51. doi: 10.1038/nrg1894 16755287

11. Jaeken J, Matthijs G. Congenital Disorders of Glycosylation: A Rapidly Expanding Disease Family. Annu Rev Genomics Hum Genet [Internet]. 2007;8(1):261–78. Available from:

12. Jeff G, Schnapp BJ, Sheetz MP. 198 8 Nature Publishing Group. Nature. 1988;331:450. doi: 10.1038/331450a0 3123999

13. Audfray A, Varrot A, Imberty A. Bacteria love our sugars: Interaction between soluble lectins and human fucosylated glycans, structures, thermodynamics and design of competing glycocompounds. Comptes Rendus Chim [Internet]. Academie des sciences; 2013;16(5):482–90. Available from:

14. Imberty A, Varrot A. Microbial recognition of human cell surface glycoconjugates. Curr Opin Struct Biol. 2008;18(5):567–76. doi: 10.1016/ 18809496

15. Dennis JW, Nabi IR, Demetriou M. Organization, Cell Surface and disease. Cell. 2009;139(7):1229–41. doi: 10.1016/j.cell.2009.12.008 20064370

16. Takahashi M, Kizuka Y, Ohtsubo K, Gu J, Taniguchi N. Disease-associated glycans on cell surface proteins. Mol Aspects Med [Internet]. Elsevier Ltd; 2016;51:56–70. Available from: doi: 10.1016/j.mam.2016.04.008 27131428

17. Taniguchi N, Kizuka Y. Glycans and cancer: Role of N-Glycans in cancer biomarker, progression and metastasis, and therapeutics [Internet]. 1st ed. Vol. 126, Advances in Cancer Research. Elsevier Inc.; 2015. 11–51 p. Available from: doi: 10.1016/bs.acr.2014.11.001 25727145

18. Kizuka Y, Kitazume S, Taniguchi N. N-glycan and Alzheimer’s disease. Biochim Biophys Acta—Gen Subj [Internet]. Elsevier B.V.; 2017;1861(10):2447–54. Available from: doi: 10.1016/j.bbagen.2017.04.012 28465241

19. Higel F, Seidl A, Sörgel F, Friess W. N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. Eur J Pharm Biopharm [Internet]. Elsevier B.V.; 2016;100(January):94–100. Available from:

20. Zhang P, Woen S, Wang T, Liau B, Zhao S, Chen C, et al. Challenges of glycosylation analysis and control: An integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today [Internet]. Elsevier Ltd; 2016;21(5):740–65. Available from: doi: 10.1016/j.drudis.2016.01.006 26821133

21. Griebenow KAI, Sola RJ. Effects of Glycosylation on the Stability of Protein Pharmaceuticals. J Pharm Sci. 2009;98(4):1223–45. doi: 10.1002/jps.21504 18661536

22. Mariño K, Bones J, Kattla JJ, Rudd PM. A systematic approach to protein glycosylation analysis: A path through the maze. Nat Chem Biol [Internet]. Nature Publishing Group; 2010;6(10):713–23. Available from: doi: 10.1038/nchembio.437 20852609

23. Balog CIA, Stavenhagen K, Fung WLJ, Koeleman CA, McDonnell LA, Verhoeven A, et al. N -glycosylation of Colorectal Cancer Tissues. Mol Cell Proteomics [Internet]. 2012;11(9):571–85. Available from: doi: 10.1074/mcp.M111.011601 22573871

24. Cornelissen LAM, Van Vliet SJ. A bitter sweet symphony: Immune responses to altered o-glycan epitopes in cancer. Biomolecules. 2016;6(2):1–19.

25. Chik JHL, Zhou J, Moh ESX, Christopherson R, Clarke SJ, Molloy MP, et al. Comprehensive glycomics comparison between colon cancer cell cultures and tumours: Implications for biomarker studies. J Proteomics [Internet]. Elsevier B.V.; 2014;108:146–62. Available from: doi: 10.1016/j.jprot.2014.05.002 24840470

26. Merry T, Astrautsova S. Chemical and Enzymatic Release of Glycans from Glycoproteins. Capill Electrophor Carbohydrates [Internet]. 213:27–40. Available from:

27. Sun G, Yu X, Bao C, Wang L, Li M, Gan J, et al. Identification and characterization of a novel prokaryotic peptide: N-Glycosidase from Elizabethkingia meningoseptica. J Biol Chem. 2015;290(12):7452–62. doi: 10.1074/jbc.M114.605493 25614628

28. Burnina I, Hoyt E, Lynaugh H, Li H, Gong B. A cost-effective plate-based sample preparation for antibody N-glycan analysis. J Chromatogr A [Internet]. Elsevier B.V.; 2013;1307:201–6. Available from: doi: 10.1016/j.chroma.2013.07.104 23932029

29. Holst S, Deuss AJM, van Pelt GW, van Vliet SJ, Garcia-Vallejo JJ, Koeleman CAM, et al. N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression. Mol Cell Proteomics [Internet]. 2016;15(1):124–40. Available from: doi: 10.1074/mcp.M115.051235 26537799

30. Jensen PH, Karlsson NG, Kolarich D, Packer NH. Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc [Internet]. Nature Publishing Group; 2012;7(7):1299–310. Available from: doi: 10.1038/nprot.2012.063 22678433

31. Jiménez-castells C, Vanbeselaere J, Kohlhuber S. Europe PMC Funders Group Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum. 2018;1861(2):418–30.

32. Inka B, Royle L, Radcliffe CM, Dwek RA, Rudd PM. Detailed Structural Analysis of N-Glycans Released From Glycoproteins in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase Array Digestions. Glycobiol Protoc. 2006;347:125–44.

33. Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM. GlycoWorkbench: A tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res. 2008;7(4):1650–9. doi: 10.1021/pr7008252 18311910

34. Varki A, Cummings RD, Aebi M, Packer NH, Seeberger PH, Esko JD, et al. Symbol nomenclature for graphical representations of glycans. Glycobiology. 2015;25(12):1323–4. doi: 10.1093/glycob/cwv091 26543186

35. Shubhakar A, Reiding KR, Gardner RA, Spencer DIR, Fernandes DL, Wuhrer M. High-Throughput Analysis and Automation for Glycomics Studies. Chromatographia. 2014;78(5–6):321–33. doi: 10.1007/s10337-014-2803-9 25814696

36. Ventham NT, Gardner RA, Kennedy NA, Shubhakar A, Kalla R, Nimmo ER, et al. Changes to serum sample tube and processing methodology does not cause inter-individual variation in automated whole serum N-Glycan profiling in health and disease. PLoS One. 2015;10(4):1–16.

37. Stöckmann H, O’Flaherty R, Adamczyk B, Saldova R, Rudd PM. Automated, high-throughput serum glycoprofiling platform. Integr Biol (United Kingdom). 2015;7(9):1026–32.

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden