Analytical and Bioanalytical Chemistry

, Volume 410, Issue 6, pp 1617–1629 | Cite as

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery

  • Jua Lee
  • Serenus Hua
  • Sung Hyeon Lee
  • Myung Jin Oh
  • Jaekyung Yun
  • Jin Young Kim
  • Jae-Han Kim
  • Jung Hoe Kim
  • Hyun Joo An
Paper in Forefront

Abstract

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography–mass spectrometry and nano liquid chromatography–tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery.

Graphical abstract

Keywords

Mass spectrometry Glycoproteomics Site-specific glycosylation Glycopeptide Gastric cancer 

Notes

Acknowledgements

This work was supported by Korea Basic Science Institute grant (T37413) and by the Institute for Basic Science (IBS-R001-D1-2017-a00).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval and consent to participat

Human sera were collected by the National Biobank of Korea by means of a standardized protocol approved by the Ethics Committees of Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea (KH2010-15). Informed consent was provided by all individuals involved in the study.

Supplementary material

216_2017_811_MOESM1_ESM.pdf (851 kb)
ESM 1 (PDF 850 kb)

References

  1. 1.
    Hart GW, Copeland RJ. Glycomics hits the big time. Cell. 2010;143(5):672–6.CrossRefGoogle Scholar
  2. 2.
    Stavenhagen K, Hinneburg H, Thaysen-Andersen M, Hartmann L, Silva DV, Fuchser J, et al. Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J Mass Spectrom. 2013;48(6):627–39.CrossRefGoogle Scholar
  3. 3.
    Ruhaak LR, H-W U, Beekman M, Hokke CH, Westendorp RG, Houwing-Duistermaat J, et al. Plasma protein N-glycan profiles are associated with calendar age, familial longevity and health. J Proteome Res. 2011;10(4):1667–74.CrossRefGoogle Scholar
  4. 4.
    Hua S, Saunders M, Dimapasoc LM, Jeong SH, Kim BJ, Kim S, et al. Differentiation of cancer cell origin and molecular subtype by plasma membrane N-glycan profiling. J Proteome Res. 2014;13(2):961–8.CrossRefGoogle Scholar
  5. 5.
    Bondt A, Selman MH, Deelder AM, Hazes JM, Willemsen SP, Wuhrer M, et al. Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation. J Proteome Res. 2013;12(10):4522–31.CrossRefGoogle Scholar
  6. 6.
    Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinical implications. Nat Rev Cancer. 2015;15(9):540–55.CrossRefGoogle Scholar
  7. 7.
    Hua S, An HJ, Ozcan S, Ro GS, Soares S, DeVere-White R, et al. Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers. Analyst. 2011;136(18):3663–71.CrossRefGoogle Scholar
  8. 8.
    Hua S, Williams CC, Dimapasoc LM, Ro GS, Ozcan S, Miyamoto S, et al. Isomer-specific chromatographic profiling yields highly sensitive and specific potential N-glycan biomarkers for epithelial ovarian cancer. J Chromatogr A. 2013;1279:58–67.  https://doi.org/10.1016/j.chroma.2012.12.079 CrossRefGoogle Scholar
  9. 9.
    Ozcan S, Barkauskas DA, Ruhaak LR, Torres J, Cooke CL, An HJ, et al. Serum glycan signatures of gastric cancer. Cancer Prev Res. 2014;7(2):226–35.CrossRefGoogle Scholar
  10. 10.
    Lee SH, Jeong S, Lee J, Yeo IS, MJ O, Kim U, et al. Glycomic profiling of targeted serum haptoglobin for gastric cancer using nano LC/MS and LC/MS/MS. Mol Biosyst. 2016;12(12):3611–21.CrossRefGoogle Scholar
  11. 11.
    Campos D, Freitas D, Gomes J, Magalhães A, Steentoft C, Gomes C, et al. Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery. Mol Cell Proteomics. 2015;14(6):1616–29.CrossRefGoogle Scholar
  12. 12.
    Shah P, Wang X, Yang W, Eshghi ST, Sun S, Hoti N, et al. Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveal glycoprotein alteration in protein abundance and glycosylation. Mol Cell Proteomics. 2015;14(10):2753–63.CrossRefGoogle Scholar
  13. 13.
    Hua S, An HJ. Glycoscience aids in biomarker discovery. BMB Rep. 2012;45(6):323–30.CrossRefGoogle Scholar
  14. 14.
    Matsuda A, Kuno A, Matsuzaki H, Kawamoto T, Shikanai T, Nakanuma Y, et al. Glycoproteomics-based cancer marker discovery adopting dual enrichment with Wisteria floribunda agglutinin for high specific glyco-diagnosis of cholangiocarcinoma. J Proteomics. 2013;85:1–11.CrossRefGoogle Scholar
  15. 15.
    Kaji H, Ocho M, Togayachi A, Kuno A, Sogabe M, Ohkura T, et al. Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma. J Proteome Res. 2013;12(6):2630–40.CrossRefGoogle Scholar
  16. 16.
    Shang S, Li W, Qin X, Zhang S, Liu Y. Aided diagnosis of hepatocellular carcinoma using serum fucosylated haptoglobin ratios. J Cancer. 2017;8(5):887.CrossRefGoogle Scholar
  17. 17.
    Sun S, Wang Q, Zhao F, Chen W, Li Z. Glycosylation site alteration in the evolution of influenza A (H1N1) viruses. PLoS One. 2011;6(7):e22844.CrossRefGoogle Scholar
  18. 18.
    Balla-Jhagjhoorsingh SS, Corti D, Heyndrickx L, Willems E, Vereecken K, Davis D, et al. The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody. PLoS One. 2013;8(7):e68863.CrossRefGoogle Scholar
  19. 19.
    Thaysen-Andersen M, Packer NH. Advances in LC–MS/MS-based glycoproteomics: getting closer to system-wide site-specific mapping of the N-and O-glycoproteome. Biochim Biophys Acta. 2014;1844(9):1437–52.CrossRefGoogle Scholar
  20. 20.
    Dallas DC, Martin WF, Hua S, German JB. Automated glycopeptide analysis—review of current state and future directions. Brief Bioinform. 2012;14(3):361–74.CrossRefGoogle Scholar
  21. 21.
    Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends—an update. Cancer Epidemiol Biomark Prev. 2016;25(1):16–27.CrossRefGoogle Scholar
  22. 22.
    Miki K, Morita M, Sasajima M, Hoshina R, Kanda E, Urita Y. Usefulness of gastric cancer screening using the serum pepsinogen test method. Am J Gastroenterol. 2003;98(4):735–9.CrossRefGoogle Scholar
  23. 23.
    Shimoyama T, Aoki M, Sasaki Y, Matsuzaka M, Nakaji S, Fukuda SABC. screening for gastric cancer is not applicable in a Japanese population with high prevalence of atrophic gastritis. Gastric Cancer. 2012;15(3):331–4.CrossRefGoogle Scholar
  24. 24.
    Chung SJ, Park MJ, Kang SJ, Kang HY, Chung GE, Kim SG, et al. Effect of annual endoscopic screening on clinicopathologic characteristics and treatment modality of gastric cancer in a high-incidence region of Korea. Int J Cancer. 2012;131(10):2376–84.CrossRefGoogle Scholar
  25. 25.
    Kim BJ, Heo C, Kim BK, Kim JY, Kim JG. Effectiveness of gastric cancer screening programs in South Korea: organized vs opportunistic models. World J Gastroenterol. 2013;19(5):736.CrossRefGoogle Scholar
  26. 26.
    Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem. 1996;42(10):1589–600.Google Scholar
  27. 27.
    Sadrzadeh SM, Saffari Y, Bozorgmehr J. Haptoglobin phenotypes in epilepsy. Clin Chem. 2004;50(6):1095–7.  https://doi.org/10.1373/clinchem.2003.028001 CrossRefGoogle Scholar
  28. 28.
    Zhang S, Shang S, Li W, Qin X, Liu Y. Insights on N-glycosylation of human haptoglobin and its association with cancers. Glycobiology. 2016;26(7):684–92.CrossRefGoogle Scholar
  29. 29.
    Kim J-H, Lee SH, Choi S, Kim U, Yeo IS, Kim SH, et al. Direct analysis of aberrant glycosylation on haptoglobin in patients with gastric cancer. Oncotarget. 2017;8(7):11094.Google Scholar
  30. 30.
    Hua S, Hu CY, Kim BJ, Totten SM, Oh MJ, Yun N, et al. Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative glycoproteomic characterization. J Proteome Res. 2013;12(10):4414-4423. https://doi.org/10.1021/pr400442y.Google Scholar
  31. 31.
    ZL W, Ethen C, Hickey GE, Jiang W. Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion. Biochem Biophys Res Commun. 2009;379(3):749–53.CrossRefGoogle Scholar
  32. 32.
    An HJ, Tillinghast JS, Woodruff DL, Rocke DM, Lebrilla CB. A new computer program (GlycoX) to determine simultaneously the glycosylation sites and oligosaccharide heterogeneity of glycoproteins. J Proteome Res. 2006;5(10):2800–8.CrossRefGoogle Scholar
  33. 33.
    Strum JS, Nwosu CC, Hua S, Kronewitter SR, Seipert RR, Bachelor RJ, et al. Automated assignments of N-and O-site specific glycosylation with extensive glycan heterogeneity of glycoprotein mixtures. Anal Chem. 2013;85(12):5666–75.CrossRefGoogle Scholar
  34. 34.
    Kronewitter SR, An HJ, De Leoz ML, Lebrilla CB, Miyamoto S, Leiserowitz GS. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome. Proteomics. 2009;9(11):2986–94.CrossRefGoogle Scholar
  35. 35.
    Hua S, MJ O, Ozcan S, Seo YS, Grimm R, An HJ. Technologies for glycomic characterization of biopharmaceutical erythropoietins. Trends Anal Chem. 2015;68:18–27.CrossRefGoogle Scholar
  36. 36.
    Stavenhagen K, Kolarich D, Wuhrer M. Clinical glycomics employing graphitized carbon liquid chromatography–mass spectrometry. Chromatographia. 2015;78(5-6):307–20.CrossRefGoogle Scholar
  37. 37.
    An HJ, Peavy TR, Hedrick JL, Lebrilla CB. Determination of N-glycosylation sites and site heterogeneity in glycoproteins. Anal Chem. 2003;75(20):5628–37.CrossRefGoogle Scholar
  38. 38.
    Juhasz P, Martin SA. The utility of nonspecific proteases in the characterization of glycoproteins by high-resolution time-of-flight mass spectrometry. Int J Mass Spectrom Ion Processes. 1997;169:217–30.CrossRefGoogle Scholar
  39. 39.
    Cancilla MT, Wong AW, Voss LR, Lebrilla CB. Fragmentation reactions in the mass spectrometry analysis of neutral oligosaccharides. Anal Chem. 1999;71(15):3206–18.CrossRefGoogle Scholar
  40. 40.
    Buck CA, Glick MC, Warren L. Glycopeptides from the surface of control and virus-transformed cells. Science. 1971;172(3979):169–71.CrossRefGoogle Scholar
  41. 41.
    Bresalier RS, Byrd JC, Tessler D, Lebel J, Koomen J, Hawke D, et al. A circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated in individuals with colon cancer. Gastroenterology. 2004;127(3):741–8.CrossRefGoogle Scholar
  42. 42.
    Sharpe-Timms KL, Zimmer RL, Ricke EA, Piva M, Horowitz GM. Endometriotic haptoglobin binds to peritoneal macrophages and alters their function in women with endometriosis. Fertil Steril. 2002;78(4):810–9.CrossRefGoogle Scholar
  43. 43.
    Park S-Y, Lee S-H, Kawasaki N, Itoh S, Kang K, Ryu SH, et al. α1-3/4 fucosylation at Asn 241 of β-haptoglobin is a novel marker for colon cancer: a combinatorial approach for development of glycan biomarkers. Int J Cancer. 2012;130(10):2366–76.CrossRefGoogle Scholar
  44. 44.
    Park S-Y, Yoon S-J, Jeong Y-T, Kim J-M, Kim J-Y, Bernert B, et al. N-glycosylation status of β-haptoglobin in sera of patients with colon cancer, chronic inflammatory diseases and normal subjects. Int J Cancer. 2010;126(1):142–55.  https://doi.org/10.1002/ijc.24685 CrossRefGoogle Scholar
  45. 45.
    Carlsson MC, Cederfur C, Schaar V, Balog CI, Lepur A, Touret F, et al. Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients. PLoS One. 2011;6(10):e26560.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Jua Lee
    • 1
    • 2
  • Serenus Hua
    • 1
    • 2
  • Sung Hyeon Lee
    • 3
  • Myung Jin Oh
    • 1
    • 2
  • Jaekyung Yun
    • 1
    • 2
  • Jin Young Kim
    • 4
  • Jae-Han Kim
    • 5
  • Jung Hoe Kim
    • 6
  • Hyun Joo An
    • 1
    • 2
  1. 1.Asia Glycomics Reference SiteChungnam National UniversityDaejeonRepublic of Korea
  2. 2.Graduate School of Analytical Science and Technology, #455 College of Engineering IIChungnam National UniversityDaejeonRepublic of Korea
  3. 3.GLYCAN Co. Ltd.Healthcare Innovation ParkSeongnamRepublic of Korea
  4. 4.Department of Mass SpectrometryKorea Basic Science InstituteOchangRepublic of Korea
  5. 5.Department of Food and NutritionChungnam National UniversityDaejeonRepublic of Korea
  6. 6.Department of Biological SciencesKorea Advanced Institute of Science and TechnologyDaejeonRepublic of Korea

Personalised recommendations