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N-Glycosylation Analysis by HPAEC-PAD and Mass Spectrometry

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Ovarian Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1049))

Abstract

Changes in protein glycosylation are a hallmark of most types of cancer including ovarian carcinoma. The structural elucidation of glycans is technically challenging and it requires complementary chromatographic and spectroscopic techniques among others. Here, we describe the profiling of N-glycans from glycoproteins of SKOV3 ovarian carcinoma cells by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS). Mass spectrometry as a complementary method enables precise mass determination of N-glycan mixtures thus corroborating data obtained from HPAEC-PAD mapping in conjunction with reference oligosaccharide structures.

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References

  1. Kulasingam V, Pavlou MP, Diamandis EP (2010) Integrating high-throughput technologies in the quest for effective biomarkers for ovarian cancer. Nat Rev Cancer 10:371–378

    Article  PubMed  CAS  Google Scholar 

  2. Abbott KL, Nairn AV, Hall EM et al (2008) Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer. Proteomics 8:3210–3220

    Article  PubMed  CAS  Google Scholar 

  3. Townsend RR, Hardy MR, Lee YC (1989) Separation of oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection. Methods Enzymol 179:65–76

    Article  PubMed  CAS  Google Scholar 

  4. Montreuil J, Bouquelet S, Debray H et al (2003) Glycoproteins. In: Chaplin MF, Kennedy JF (eds) Carbohydrate analysis - a practical approach, 2nd edn. Oxford, Oxford University Press, pp 181–293

    Google Scholar 

  5. Morelle W, Michalski JC (2005) The mass spectrometric analysis of glycoproteins and their glycan structures. Curr Anal Chem 1:29–57

    Article  CAS  Google Scholar 

  6. Sutton-Smith M, Dell A (2006) Analysis of carbohydrates/glycoproteins by mass spectrometry. In: Celis JE, Carter N, Simons K, Small JV, Hunter T, Shotton D (eds) Cell biology, volume 4, 3rd edn. Burlington, San Diego, London, Elsevier Science, p 415–425

    Google Scholar 

  7. Harvey DJ (2011) Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for the period 2005–2006. Mass Spectrom Rev 30:1–100

    Article  PubMed  CAS  Google Scholar 

  8. Hillenkamp F, Karas M (1990) Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol 193:280–295

    Article  PubMed  CAS  Google Scholar 

  9. Pohl S, Hoffmann A, Rudiger A et al (1997) Hypoglycosylation of a brain glycoprotein (beta-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency. Glycobiology 7:1077–1084

    Article  PubMed  CAS  Google Scholar 

  10. Machado E, Kandzia S, Carilho R et al (2011) N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin. Glycobiology 21:376–386

    Article  PubMed  CAS  Google Scholar 

  11. Brito C, Kandzia S, Graca T et al (2008) Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization. Biochimie 90:1279–1290

    Article  PubMed  CAS  Google Scholar 

  12. Maass K, Ranzinger R, Geyer H et al (2007) "Glyco-peakfinder"–de novo composition analysis of glycoconjugates. Proteomics 7:4435–4444

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We acknowledge Fundação para a Ciência e a Tecnologia, Portugal, projects PTDC/SAU-NEU/100724/2008 and PIC/IC/82765/2007.

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Kandzia, S., Costa, J. (2013). N-Glycosylation Analysis by HPAEC-PAD and Mass Spectrometry. In: Malek, A., Tchernitsa, O. (eds) Ovarian Cancer. Methods in Molecular Biology, vol 1049. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-547-7_22

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  • DOI: https://doi.org/10.1007/978-1-62703-547-7_22

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-546-0

  • Online ISBN: 978-1-62703-547-7

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