Analytical and Bioanalytical Chemistry

, Volume 407, Issue 7, pp 1857–1869 | Cite as

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

  • Xue Bai
  • Daoyuan Li
  • Jing Zhu
  • Yudong Guan
  • Qunye Zhang
  • Lianli Chi
Research Paper
First Online:
Received:
Accepted:

Abstract

O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography–tandem mass spectrometry (LC–MS–MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC–MS–MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.

Keywords

O-glycosylation site hCG Human-plasma proteins Mass spectrometry Exoglycosidases Jacalin 
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Notes

Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973 Program) (2012CB822102), the National High Technology Research and Development Program of China (863 Program) (2012AA021504), the National Natural Science Foundation of China (21472115, 81102783, 31000367), and Natural Science Foundation of Shandong Province, China (ZR2011HQ038).

Supplementary material

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References

  1. 1.
    Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA (2001) Glycosylation and the immune system. Science 291:2370–2376CrossRefGoogle Scholar
  2. 2.
    Helenius A, Aebi M (2001) Intracellular functions of N-linked glycans. Science 291:2364–2369CrossRefGoogle Scholar
  3. 3.
    Konopka JB (2012) N-acetylglucosamine (GlcNAc) functions in cell signaling. Scientifica (Cairo)Google Scholar
  4. 4.
    Fardini Y, Dehennaut V, Lefebvre T, Issad T (2013) O-GlcNAcylation: a new cancer hallmark? Front Endocrinol (Lausanne) 4:99Google Scholar
  5. 5.
    Whitmore TE, Peterson A, Holzman T, Eastham A, Amon L, McIntosh M, Ozinsky A, Nelson PS, Martin DB (2012) Integrative analysis of N-linked human glycoproteomic data sets reveals PTPRF ectodomain as a novel plasma biomarker candidate for prostate cancer. J Proteome Res 11:2653–2665CrossRefGoogle Scholar
  6. 6.
    Yao PJ, Coleman PD (1998) Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer’s disease. J Neurosci 18:2399–2411Google Scholar
  7. 7.
    Huang Y, Wu H, Xue R, Liu T, Dong L, Yao J, Zhang Y, Shen X (2013) Identification of N-glycosylation in hepatocellular carcinoma patients' serum with a comparative proteomic approach. PLoS One 8:e77161CrossRefGoogle Scholar
  8. 8.
    Dunfee RL, Thomas ER, Wang J, Kunstman K, Wolinsky SM, Gabuzda D (2007) Loss of the N-linked glycosylation site at position 386 in the HIV envelope V4 region enhances macrophage tropism and is associated with dementia. Virology 367:222–234CrossRefGoogle Scholar
  9. 9.
    Yamamoto K (1994) Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins. J Biochem 116:229–235Google Scholar
  10. 10.
    Narimatsu H, Sawaki H, Kuno A, Kaji H, Ito H, Ikehara Y (2010) A strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics. FEBS J 277:95–105CrossRefGoogle Scholar
  11. 11.
    Davies MJ, Smith KD, Hounsell EF (1994) In: Walker M (ed) Methods in molecular biology: basic protein and peptide protocols, 2nd edn. Humana Press, TotowaGoogle Scholar
  12. 12.
    Chiesa C, O’Neill RA, Horváth CG, Oefner PJ (1996) In: Righetti PG (ed) Capillary electrophoresis in analytical biotechnology. CRC Press, Boca RatonGoogle Scholar
  13. 13.
    Rademaker GJ, Pergantis SA, Blok-Tip L, Langridge JI, Kleen A, Thomas-Oates JE (1998) Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity. Anal Biochem 257:149–160CrossRefGoogle Scholar
  14. 14.
    Zheng Y, Guo Z, Cai Z (2009) Combination of beta-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of O-glycosylation sites. Talanta 78:358–363CrossRefGoogle Scholar
  15. 15.
    Hanisch FG, Teitz S, Schwientek T, Müller S (2009) Chemical de-O-glycosylation of glycoproteins for application in LC-based proteomics. Proteomics 9:710–719CrossRefGoogle Scholar
  16. 16.
    Whitaker JR, Feeney RE (1977) Behavior of O-glycosyl and O-phosphoryl proteins in alkaline solution. Adv Exp Med Biol 86:155–175CrossRefGoogle Scholar
  17. 17.
    Hägglund P, Matthiesen R, Elortza F, Højrup P, Roepstorff P, Jensen ON, Bunkenborg J (2007) An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins. J Proteome Res 6:3021–3031CrossRefGoogle Scholar
  18. 18.
    Steentoft C, Vakhrushev SY, Vester-Christensen MB, Schjoldager KT, Kong Y, Bennett EP, Mandel U, Wandall H, Levery SB, Clausen H (2011) Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nat Methods 8:977–982CrossRefGoogle Scholar
  19. 19.
    Ongay S, Boichenko A, Govorukhina N, Bischoff R (2012) Glycopeptide enrichment and separation for protein glycosylation analysis. J Sep Sci 35:2341–2372CrossRefGoogle Scholar
  20. 20.
    Pan S, Chen R, Aebersold R, Brentnall TA (2011) Mass spectrometry based glycoproteomics–from a proteomics perspective. Mol Cell Proteomics 10:R110.003251CrossRefGoogle Scholar
  21. 21.
    Tachibana K, Nakamura S, Wang H, Iwasaki H, Tachibana K, Maebara K, Cheng L, Hirabayashi J, Narimatsu H (2006) Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography. Glycobiology 16:46–53CrossRefGoogle Scholar
  22. 22.
    Saroha A, Kumar S, Chatterjee BP, Das HR (2012) Jacalin bound plasma O-glycoproteome and reduced sialylation of alpha 2-HS glycoprotein (A2HSG) in rheumatoid arthritis patients. PLoS One 7:e46374CrossRefGoogle Scholar
  23. 23.
    Darula Z, Medzihradszky KF (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol Cell Proteomics 8:2515–2526CrossRefGoogle Scholar
  24. 24.
    Darula Z, Sherman J, Medzihradszky KF (2012) How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides. Mol Cell Proteomics 11:O111.016774CrossRefGoogle Scholar
  25. 25.
    Nilsson J, Rüetschi U, Halim A, Hesse C, Carlsohn E, Brinkmalm G, Larson G (2009) Enrichment of glycopeptides for glycan structure and attachment site identification. Nat Methods 6:809–811CrossRefGoogle Scholar
  26. 26.
    Halim A, Nilsson J, Rüetschi U, Hesse C, Larson G (2012) Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD. Mol Cell Proteomics 11:M111.013649CrossRefGoogle Scholar
  27. 27.
    Halim A, Rüetschi U, Larson G, Nilsson J (2013) LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins. J Proteome Res 12:573–584CrossRefGoogle Scholar
  28. 28.
    Zauner G, Koeleman CA, Deelder AM, Wuhrer M (2010) Protein glycosylation analysis by HILIC-LC-MS of proteinase K-generated N- and O-glycopeptides. J Sep Sci 33:903–910CrossRefGoogle Scholar
  29. 29.
    Nwosu CC, Seipert RR, Strum JS, Hua SS, An HJ, Zivkovic AM, German BJ, Lebrilla CB (2011) Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures. J Proteome Res 10:2612–2624CrossRefGoogle Scholar
  30. 30.
    Durham M, Regnier FE (2006) Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome. J Chromatogr A 1132:165–173CrossRefGoogle Scholar
  31. 31.
    Kessler MJ, Mise T, Ghai RD, Bahl OP (1979) Structure and location of the O-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem 254:7909–7914Google Scholar
  32. 32.
    Morgan FJ, Birken S, Canfield RE (1975) The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit. J Biol Chem 250:5247–5258Google Scholar
  33. 33.
    Valmu L, Alfthan H, Hotakainen K, Birken S, Stenman UH (2006) Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography–electrospray mass spectrometry. Glycobiology 16:1207–1218CrossRefGoogle Scholar
  34. 34.
    Bourne Y, Astoul CH, Zamboni V, Peumans WJ, Menu-Bouaouiche L, Van Damme EJ, Barre A, Rougé P (2002) Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose. Biochem J 364:173–180Google Scholar
  35. 35.
    Pisano A, Jardine DR, Packer NH, Farnsworth V, Carson W, Cartier P, Redmond JW, Williams KL, Gooley AA (1996) In: Townsend RR, Hotchkiss AT Jr (eds) Techniques in glycobiology. Marcel Dekker, New YorkGoogle Scholar
  36. 36.
    Wiesner J, Premsler T, Sickmann A (2008) Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications. Proteomics 8:4466–4483CrossRefGoogle Scholar
  37. 37.
    Cole LA, Butler S (2012) Hyperglycosylated hCG, hCGβ and hyperglycosylated hCGβ: interchangeable cancer promoters. Mol Cell Endocrinol 349:232–238CrossRefGoogle Scholar
  38. 38.
    Cole LA, Laidler LL, Muller CY (2010) USA hCG reference service, 10-year report. Clin Biochem 43:1013–1022CrossRefGoogle Scholar
  39. 39.
    Cole LA (2009) Human chorionic gonadotropin and associated molecules. Expert Rev Mol Diagn 9:51–73CrossRefGoogle Scholar
  40. 40.
    Cole LA (2010) Hyperglycosylated hCG, a review. Placenta 31:653–664CrossRefGoogle Scholar
  41. 41.
    Zhao X, Ma C, Han H, Jiang J, Tian F, Wang J, Ying W, Qian X (2013) Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment. Anal Bioanal Chem 405:5519–5529CrossRefGoogle Scholar
  42. 42.
    Zauner G, Hoffmann M, Rapp E, Koeleman CA, Dragan I, Deelder AM, Wuhrer M, Hensbergen PJ (2012) Glycoproteomic analysis of human fibrinogen reveals novel regions of O-glycosylation. J Proteome Res 11:5804–5814Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Xue Bai
    • 1
  • Daoyuan Li
    • 2
  • Jing Zhu
    • 2
  • Yudong Guan
    • 1
  • Qunye Zhang
    • 3
  • Lianli Chi
    • 1
    • 2
  1. 1.National Glycoengineering Research CenterShandong UniversityJinanChina
  2. 2.State Key Laboratory of Microbial TechnologyShandong UniversityJinanChina
  3. 3.Key Laboratory of Cardiovascular Remodeling and Function ResearchQilu Hospital of Shandong UniversityJinanChina

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