Glycoconjugate Journal

, Volume 36, Issue 1, pp 57–68 | Cite as

Identification a novel clinical biomarker in early diagnosis of human non-small cell lung cancer

  • Yanxia Jin
  • Yajun Yang
  • Yanting Su
  • Xiangdong Ye
  • Wei Liu
  • Qing Yang
  • Jie Wang
  • Xiangning Fu
  • Yongsheng GongEmail author
  • Hui SunEmail author
Original Article


Non-small cell lung cancer (NSCLC) is a malignant tumor with high morbidity and mortality. The clinical biomarkers currently used for the early diagnosis of lung cancer have poor sensitivity and specificity. Therefore, it is urgent to identify sensitive biomarkers for the early detection of NSCLC to improve the patient survival of patients. In our previously study, we identified glycoprotein alpha-1-antichymotrypsin (AACT) as an early biomarker of NSCLC. In this study, serum glycopeptides were enriched using the high-GlcNAc-specific binding lectin, AANL/AAL2, for further quantitative proteomics analysis using LC-MS/MS. A total of 55 differentially expressed proteins were identified by using demethylation labelling proteomics. Serum paraoxonase/arylesterase 1 (PON1) was selected for validation by western blotting and lectin-ELISA in samples from 120 enrolled patients. Our data showed that AANL-enriched PON1 has better diagnostic performance than total PON1 in early NSCLC, since it differed between early Stage I tumor samples and tumor-free samples (healthy and benign). Combining AANL-enriched PON1 with carcinoembryonic antigen (CEA) significantly improved the diagnostic specificity of CEA. Moreover, combined AANL-enriched PON1 and AANL-enriched AACT was significantly different between early NSCLC samples and tumor-free samples with an AUC of 0.940, 94.4% sensitivity, and 90.2% specificity. Our findings suggest that combined AANL-enriched PON1 and AANL-enriched AACT is a potential clinical biomarker for the early diagnosis of NSCLC.


Non-small cell lung cancer Biomarker Dimethylation labeling Lectin AANL Serum paraoxonase/arylesterase 1 Alpha-1-antichymotrypsin 



non-small cell lung cancer




squamous cell carcinoma


tumour node metastasis




agrocybe aegerita lectin 2 (high affinity to GlcNAc)




gene ontology

PNGase F

peptide N-glycosidase F


horse radish peroxidase


serum paraoxonase/arylesterase 1




receiver operator characteristic


area under curve


enzyme-linked immunosorbent assay


carcinoembryonic antigen


bovine serum albumin


mass spectrum


differentially expressed protein


healthy and benign



We would like to thank Professor Xiangdong Fu (University of California, San Diego, USA) for his helpful suggestions on the study. We thank Pengfei Cheng in Wuhan University Hospital and Wangjie in Tongji Medical Hospital for collection of serum samples. We thank Haoyu Li, Feilong Yu and Xi Chen for protein labeling and analysis of MS data. This work was supported by the Natural Science Foundation of China (NSFC) program [grant numbers No. 81670531, 31370849, 31800676], Jiangsu Natural Science Foundation program (grant number No. BK20141176), the Chinese 111 project [grant number No. B06018], and Hong Kong Scholars Program (grant number No. XJ2018060).

Author’s contribution

YX. Jin designed the experiments, performed most experiments, analyzed the data, and wrote the manuscript. YJ. Yang performed the western blot experiments with assistance from YX. Jin. YT. Su and XD. Ye contributed to enrichment with lectin and lectin-elisa. W. Liu contribute to provide the PNGase F enzyme. Q. Yang contribute to provide the lectin AAL2. J. Wang and XN. Fu contributed to clinical patient information management and provided all clinical patient serum samples. H. Sun and YS. Gong provided overall project supervision and revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors have stated that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

10719_2018_9853_MOESM1_ESM.pdf (213 kb)
ESM 1 (PDF 213 kb)


  1. 1.
    Parkin, D.M., Bray, F., Ferlay, J., Pisani, P.: Global cancer statistics, 2002. CA Cancer J. Clin. 55(2), 74–108 (2005)CrossRefGoogle Scholar
  2. 2.
    Akhtar-Danesh, N., Finley, C.: Temporal trends in the incidence and relative survival of non-small cell lung cancer in Canada: a population-based study. Lung Cancer. 90(1), 8–14 (2015)CrossRefGoogle Scholar
  3. 3.
    Wallace, W.A.: The challenge of classifying poorly differentiated tumours in the lung. Histopathology. 54(1), 28–42 (2009)CrossRefGoogle Scholar
  4. 4.
    Cane, P., Linklater, K.M., Nicholson, A.G., Peake, M.D., Gosney, J.: Morphological and genetic classification of lung cancer: variation in practice and implications for tailored treatment. Histopathology. 67(2), 216–224 (2015)CrossRefGoogle Scholar
  5. 5.
    Iwama, E., Okamoto, I., Yabuuchi, H., Takayama, K., Harada, T., Matsuo, Y., Tokunaga, S., Baba, E., Nakanishi, Y.: Characteristics of smoking patients with lung Cancer with emphysematous bullae. J. Thorac. Oncol. 11(9), 1586–1590 (2016)CrossRefGoogle Scholar
  6. 6.
    Brambilla, E., Travis, W.D., Colby, T.V., Corrin, B., Shimosato, Y.: The new World Health Organization classification of lung tumours. Eur. Respir. J. 18(6), 1059–1068 (2001)CrossRefGoogle Scholar
  7. 7.
    Devarakonda, S., Morgensztern, D., Govindan, R.: Genomic alterations in lung adenocarcinoma. The lancet oncology. 16(7), e342–e351 (2015)CrossRefGoogle Scholar
  8. 8.
    Pan, Y., Wang, R., Ye, T., Li, C., Hu, H., Yu, Y., Zhang, Y., Wang, L., Luo, X., Li, H., Li, Y., Shen, L., Sun, Y., Chen, H.: Comprehensive analysis of oncogenic mutations in lung squamous cell carcinoma with minor glandular component. Chest. 145(3), 473–479 (2014)CrossRefGoogle Scholar
  9. 9.
    Hoffman, P.C., Mauer, A.M., Vokes, E.E.: Lung cancer. Lancet. 355(9202), 479–485 (2000)CrossRefGoogle Scholar
  10. 10.
    Ghosal, R., Kloer, P., Lewis, K.E.: A review of novel biological tools used in screening for the early detection of lung cancer. Postgrad. Med. J. 85(1005), 358–363 (2009)CrossRefGoogle Scholar
  11. 11.
    Mulshine, J.L., Sullivan, D.C.: Clinical practice. Lung cancer screening. New Engl. J. Med. 352(26), 2714–2720 (2005)CrossRefGoogle Scholar
  12. 12.
    Hassanein, M., Callison, J.C., Callaway-Lane, C., Aldrich, M.C., Grogan, E.L., Massion, P.P.: The state of molecular biomarkers for the early detection of lung cancer. Cancer Prev. Res. 5(8), 992–1006 (2012)CrossRefGoogle Scholar
  13. 13.
    Ferrigno, D., Buccheri, G., Biggi, A.: Serum tumour markers in lung cancer: history, biology and clinical applications. Eur. Respir. J. 7(1), 186–197 (1994)CrossRefGoogle Scholar
  14. 14.
    Molina, R., Filella, X.: Aug, eacute, M., J., Fuentes, R., Bover, I., Rifa, J., Moreno, V., canals, E., vi, ntilde, olas, N., Marquez, a., Barreiro, E., Borras, J., Viladiu, P.: tumor markers (CEA, CA 125, CYFRA 21-1, SCC and NSE) in patients with non-small cell lung Cancer as an aid in histological diagnosis and prognosis. Tumor Biol. 24(4), 209–218 (2003)CrossRefGoogle Scholar
  15. 15.
    Baek, A.R., Seo, H.J., Lee, J.H., Park, S.W., Jang, A.S., Paik, S.H., Koh, E.S., Shin, H.K., Kim, D.J.: Prognostic value of baseline carcinoembryonic antigen and cytokeratin 19 fragment levels in advanced non-small cell lung cancer. Cancer Biomark. 22, 55–62 (2018)CrossRefGoogle Scholar
  16. 16.
    Liloglou, T., Bediaga, N.G., Brown, B.R., Field, J.K., Davies, M.P.: Epigenetic biomarkers in lung cancer. Cancer Lett. 342(2), 200–212 (2014)CrossRefGoogle Scholar
  17. 17.
    Shen, S., Zhang, R., Guo, Y., Loehrer, E., Wei, Y., Zhu, Y., Yuan, Q., Moran, S., Fleischer, T., Bjaanaes, M.M., Karlsson, A., Planck, M., Staaf, J., Helland, A., Esteller, M., Su, L., Chen, F., Christiani, D.C.: A multi-omic study reveals BTG2 as a reliable prognostic marker for early-stage non-small cell lung cancer. Mol. Oncol. 12, 913–924 (2018)CrossRefGoogle Scholar
  18. 18.
    Potprommanee, L., Ma, H.T., Shank, L., Juan, Y.H., Liao, W.Y., Chen, S.T., Yu, C.J.: GM2-activator protein: a new biomarker for lung cancer. J. Thorac. Oncol. 10(1), 102–109 (2015)CrossRefGoogle Scholar
  19. 19.
    Wang, P., Yang, D., Zhang, H., Wei, X., Ma, T., Cheng, Z., Hong, Q., Hu, J., Zhuo, H., Song, Y., Jia, C., Jing, F., Jin, Q., Bai, C., Mao, H., Zhao, J.: Early detection of lung Cancer in serum by a panel of MicroRNA biomarkers. Clin Lung Cancer. 16(4), 313–319 e311 (2015)CrossRefGoogle Scholar
  20. 20.
    Indovina, P., Marcelli, E., Pentimalli, F., Tanganelli, P., Tarro, G., Giordano, A.: Mass spectrometry-based proteomics: the road to lung cancer biomarker discovery. Mass Spectrom. Rev. 32(2), 129–142 (2013)CrossRefGoogle Scholar
  21. 21.
    Ahn, J.M., Sung, H.J., Yoon, Y.H., Kim, B.G., Yang, W.S., Lee, C., Park, H.M., Kim, B.J., Lee, S.Y., An, H.J., Cho, J.Y.: Integrated glycoproteomics demonstrates fucosylated serum paraoxonase 1 alterations in small cell lung cancer. Molecular & Cellular Proteomics : MCP. 13(1), 30–48 (2014)CrossRefGoogle Scholar
  22. 22.
    Liu, Y., Wang, C., Wang, R., Wu, Y., Zhang, L., Liu, B.F., Cheng, L., Liu, X.: Isomer-specific profiling of N-glycans derived from human serum for potential biomarker discovery in pancreatic cancer. J. Proteome. 181, 160-169 (2018)Google Scholar
  23. 23.
    Wen, C.L., Chen, K.Y., Chen, C.T., Chuang, J.G., Yang, P.C., Chow, L.P.: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker. J. Proteome. 75(13), 3963–3976 (2012)CrossRefGoogle Scholar
  24. 24.
    Drake, P.M., Schilling, B., Niles, R.K., Prakobphol, A., Li, B., Jung, K., Cho, W., Braten, M., Inerowicz, H.D., Williams, K., Albertolle, M., Held, J.M., Iacovides, D., Sorensen, D.J., Griffith, O.L., Johansen, E., Zawadzka, A.M., Cusack, M.P., Allen, S., Gormley, M., Hall, S.C., Witkowska, H.E., Gray, J.W., Regnier, F., Gibson, B.W., Fisher, S.J.: Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers. J. Proteome Res. 11(4), 2508–2520 (2012)CrossRefGoogle Scholar
  25. 25.
    Satomaa, T., Heiskanen, A., Leonardsson, I., Angstrom, J., Olonen, A., Blomqvist, M., Salovuori, N., Haglund, C., Teneberg, S., Natunen, J., Carpen, O., Saarinen, J.: Analysis of the human cancer glycome identifies a novel group of tumor-associated N-acetylglucosamine glycan antigens. Cancer Res. 69(14), 5811–5819 (2009)CrossRefGoogle Scholar
  26. 26.
    Jiang, S., Chen, Y., Wang, M., Yin, Y., Pan, Y., Gu, B., Yu, G., Li, Y., Wong, B.H., Liang, Y., Sun, H.: A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem. J. 443(2), 369–378 (2012)CrossRefGoogle Scholar
  27. 27.
    Jin, Y., Wang, J., Ye, X., Su, Y., Yu, G., Yang, Q., Liu, W., Yu, W., Cai, J., Chen, X., Liang, Y., Chen, Y., Wong, B.H., Fu, X., Sun, H.: Identification of GlcNAcylated alpha-1-antichymotrypsin as an early biomarker in human non-small-cell lung cancer by quantitative proteomic analysis with two lectins. Br. J. Cancer. 114(5), 532–544 (2016)CrossRefGoogle Scholar
  28. 28.
    West-Nielsen, M., Hogdall, E.V., Marchiori, E., Hogdall, C.K., Schou, C., Heegaard, N.H.: Sample handling for mass spectrometric proteomic investigations of human sera. Anal. Chem. 77(16), 5114–5123 (2005)CrossRefGoogle Scholar
  29. 29.
    Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A.J.: Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4(4), 484–494 (2009)CrossRefGoogle Scholar
  30. 30.
    Li, L., Song, J., Kalt, W., Forney, C., Tsao, R., Pinto, D., Chisholm, K., Campbell, L., Fillmore, S., Li, X.: Quantitative proteomic investigation employing stable isotope labeling by peptide dimethylation on proteins of strawberry fruit at different ripening stages. J. Proteome. 94, 219–239 (2013)CrossRefGoogle Scholar
  31. 31.
    Zielinska, D.F., Gnad, F., Wisniewski, J.R., Mann, M.: Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell. 141(5), 897–907 (2010)CrossRefGoogle Scholar
  32. 32.
    Zeng, G.Q., Zhang, P.F., Deng, X., Yu, F.L., Li, C., Xu, Y., Yi, H., Li, M.Y., Hu, R., Zuo, J.H., Li, X.H., Wan, X.X., Qu, J.Q., He, Q.Y., Li, J.H., Ye, X., Chen, Y., Li, J.Y., Xiao, Z.Q.: Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics. Mol. Cell. Proteomics : MCP. 11(6), M111 013946 (2012)Google Scholar
  33. 33.
    Wu, J., Xie, X., Nie, S., Buckanovich, R.J., Lubman, D.M.: Altered expression of sialylated glycoproteins in ovarian cancer sera using lectin-based ELISA assay and quantitative glycoproteomics analysis. J. Proteome Res. 12(7), 3342–3352 (2013)CrossRefGoogle Scholar
  34. 34.
    Liang, Y., Ma, T., Thakur, A., Yu, H., Gao, L., Shi, P., Li, X., Ren, H., Jia, L., Zhang, S., Li, Z., Chen, M.: Differentially expressed glycosylated patterns of alpha-1-antitrypsin as serum biomarkers for the diagnosis of lung cancer. Glycobiology. 25(3), 331–340 (2015)CrossRefGoogle Scholar
  35. 35.
    Youden, W.J.: Index for rating diagnostic tests. Cancer. 3(1), 32–35 (1950)CrossRefGoogle Scholar
  36. 36.
    Elkiran, E.T., Mar, N., Aygen, B., Gursu, F., Karaoglu, A., Koca, S.: Serum paraoxonase and arylesterase activities in patients with lung cancer in a Turkish population. BMC Cancer. 7(48), (2007)Google Scholar
  37. 37.
    Dennis, J.W., Granovsky, M., Warren, C.E.: Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta. 1473(1), 21–34 (1999)CrossRefGoogle Scholar
  38. 38.
    Wu, J., Fang, M., Zhou, X., Zhu, B., Yang, Z.: Paraoxonase 1 gene polymorphisms are associated with an increased risk of breast cancer in a population of Chinese women. Oncotarget. 8(15), 25362–25371 (2017)CrossRefGoogle Scholar
  39. 39.
    Atay, A.E., Kaplan, M.A., Evliyaoglu, O., Ekin, N., Isikdogan, A.: The predictive role of Paraoxonase 1 (PON1) activity on survival in patients with metastatic and nonmetastatic gastric cancer. Clin. Ter. 165(1), e1–e5 (2014)Google Scholar
  40. 40.
    Keskin, M., Dolar, E., Dirican, M., Kiyici, M., Yilmaz, Y., Gurel, S., Nak, S.G., Erdinc, S., Gulten, M.: Baseline and salt-stimulated paraoxonase and arylesterase activities in patients with chronic liver disease: relation to disease severity. Intern. Med. J. 39(4), 243–248 (2009)CrossRefGoogle Scholar
  41. 41.
    Huang, C., Wang, Y., Liu, S., Ding, G., Liu, W., Zhou, J., Kuang, M., Ji, Y., Kondo, T., Fan, J.: Quantitative proteomic analysis identified paraoxonase 1 as a novel serum biomarker for microvascular invasion in hepatocellular carcinoma. J. Proteome Res. 12(4), 1838–1846 (2013)CrossRefGoogle Scholar
  42. 42.
    Sun, C., Chen, P., Chen, Q., Sun, L., Kang, X., Qin, X., Liu, Y.: Serum paraoxonase 1 heteroplasmon, a fucosylated, and sialylated glycoprotein in distinguishing early hepatocellular carcinoma from liver cirrhosis patients. Acta Biochim. Biophys. Sin. Shanghai. 44(9), 765–773 (2012)CrossRefGoogle Scholar
  43. 43.
    Zhang, S., Jiang, K., Zhang, Q., Guo, K., Liu, Y.: Serum fucosylated paraoxonase 1 as a potential glycobiomarker for clinical diagnosis of early hepatocellular carcinoma using ELISA index. Glycoconj. J. 32(3–4), 119–125 (2015)CrossRefGoogle Scholar
  44. 44.
    Aldonza, M.B.D., Son, Y.S., Sung, H.J., Ahn, J.M., Choi, Y.J., Kim, Y.I., Cho, S., Cho, J.Y.: Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells. Oncotarget. 8, 42817–42835 (2017)CrossRefGoogle Scholar
  45. 45.
    Sun, S., Zhang, H.: Identification and validation of atypical N-glycosylation sites. Anal. Chem. 87(24), 11948–11951 (2015)CrossRefGoogle Scholar
  46. 46.
    Lowenthal, M.S., Davis, K.S., Formolo, T., Kilpatrick, L.E., Phinney, K.W.: Identification of novel N-glycosylation sites at noncanonical protein consensus motifs. J. Proteome Res. 15(7), 2087–2101 (2016)CrossRefGoogle Scholar
  47. 47.
    Ma, C., Qu, J., Li, X., Zhao, X., Li, L., Xiao, C., Edmunds, G., Gashash, E., Song, J., Wang, P.G.: Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation. J. Proteome. 146, 90–98 (2016)CrossRefGoogle Scholar
  48. 48.
    Liu, T., Qian, W.J., Gritsenko, M.A., Camp 2nd, D.G., Monroe, M.E., Moore, R.J., Smith, R.D.: Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J. Proteome Res. 4(6), 2070–2080 (2005)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yanxia Jin
    • 1
    • 2
  • Yajun Yang
    • 1
  • Yanting Su
    • 1
  • Xiangdong Ye
    • 1
  • Wei Liu
    • 1
  • Qing Yang
    • 1
  • Jie Wang
    • 3
  • Xiangning Fu
    • 3
  • Yongsheng Gong
    • 4
    Email author
  • Hui Sun
    • 1
    • 5
    Email author
  1. 1.Hubei Key Laboratory of Cell Homeostasis, College of Life SciencesWuhan UniversityWuhanPeople’s Republic of China
  2. 2.Hubei Key Laboratory of Edible Wild Plants Conservation and UtilizationHubei Normal UniversityHuangshiPeople’s Republic of China
  3. 3.Tongji Medical HospitalHuazhong University of Science and TechnologyWuhanPeople’s Republic of China
  4. 4.Suzhou Municipal Hospitalthe Affiliated Suzhou Hospital of Nanjing Medical UniversitySuzhouPeople’s Republic of China
  5. 5.Hubei Province Key Laboratory of Allergy and ImmunologyWuhan UniversityWuhanPeople’s Republic of China

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