Skip to main content

Advertisement

SpringerLink
Log in

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

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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

ᅟ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Hart GW, Copeland RJ. Glycomics hits the big time. Cell. 2010;143(5):672–6.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  6. Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinical implications. Nat Rev Cancer. 2015;15(9):540–55.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  13. Hua S, An HJ. Glycoscience aids in biomarker discovery. BMB Rep. 2012;45(6):323–30.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  26. Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem. 1996;42(10):1589–600.

    CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. 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.

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  36. Stavenhagen K, Kolarich D, Wuhrer M. Clinical glycomics employing graphitized carbon liquid chromatography–mass spectrometry. Chromatographia. 2015;78(5-6):307–20.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  40. Buck CA, Glick MC, Warren L. Glycopeptides from the surface of control and virus-transformed cells. Science. 1971;172(3979):169–71.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Author information

Authors and Affiliations

  1. Asia Glycomics Reference Site, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 305-764, Republic of Korea

    Jua Lee, Serenus Hua, Myung Jin Oh, Jaekyung Yun & Hyun Joo An

  2. Graduate School of Analytical Science and Technology, #455 College of Engineering II, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 305-764, Republic of Korea

    Jua Lee, Serenus Hua, Myung Jin Oh, Jaekyung Yun & Hyun Joo An

  3. GLYCAN Co. Ltd., Healthcare Innovation Park, 172 Dolma-ro, Bundang-gu, Seongnam, 13605, Republic of Korea

    Sung Hyeon Lee

  4. Department of Mass Spectrometry, Korea Basic Science Institute, Ochang, 863-883, Republic of Korea

    Jin Young Kim

  5. Department of Food and Nutrition, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 305-764, Republic of Korea

    Jae-Han Kim

  6. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

    Jung Hoe Kim

Authors
  1. Jua Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Serenus Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sung Hyeon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Myung Jin Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaekyung Yun
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jin Young Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jae-Han Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jung Hoe Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hyun Joo An
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyun Joo An.

Ethics declarations

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.

Electronic supplementary material

ESM 1

(PDF 850 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, J., Hua, S., Lee, S.H. et al. Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery. Anal Bioanal Chem 410, 1617–1629 (2018). https://doi.org/10.1007/s00216-017-0811-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0811-y

Keywords

Search

Navigation