Skip to main content

In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.


Sialic acid Neu5Gc, a non-human glycan, is recognized as a new harmful substance that can cause vascular disease and cancer. Humans are unable to synthesize Neu5Gc due to a genetic defect that converts Neu5Ac to Neu5Gc, but Neu5Gc is often observed in human biological samples. Therefore, the demand for accurately measuring the amount of Neu5Gc present in human blood or tissues is rapidly increasing, but there is still no method to reliably quantify trace amounts of a non-human sugar. In particular, selective isolation and detection of Neu5Gc from human serum is analytically challenging due to the presence of excess sialic acid Neu5Ac, which has physicochemical properties very similar to Neu5Gc. Herein, we developed the label-free approach based on ZIC-HILIC/MRM-MS that can enrich sialic acids released from human serum and simultaneously monitor Neu5Ac and Neu5Gc. The combination of complete separation of Neu5Gc from abundant Neu5Ac by hydrophilic and electrostatic interactions with selective monitoring of structure-specific cross-ring cleavage ions generated by negative CID-MS/MS was remarkably effective for quantification of Neu5Ac and Neu5Gc at the femtomole level. Indeed, we were able to successfully determine the absolute quantitation of Neu5Gc from 30 healthy donors in the range of 3.336 ± 1.252 pg/μL (mean ± SD), 10,000 times lower than Neu5Ac. In particular, analysis of sialic acids in protein-free serum revealed that both Neu5Ac and Neu5G are mostly bound to proteins and/or lipids, but not in free form. In addition, the correlation between expression level of Neu5Gc and biological factors such as BMI, age, and sex was investigated. This method can be widely used in studies requiring sialic acid–related measurements such as disease diagnosis or prediction of immunogenicity in biopharmaceuticals as it is both fast and highly sensitive.

Graphical abstract

This is a preview of subscription content, access via your institution.

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



Sialic acid


N-Acetylneuraminic acid


N-Glycolylneuraminic acid


Cytidine monophospho-N-acetylneuraminic acid hydroxylase




Liquid chromatography


Mass spectrometry


Zwitterionic hydrophilic interaction liquid chromatography


Selection/multiple reaction monitoring


Collision-induced dissociation


Standard deviation


Coefficient of variation


Limited of detection


Lower limited of quantitation


Body mass index


  1. 1.

    Angata T, Varki A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev. 2002;102(2):439–69.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Traving C, Schauer R. Structure, function and metabolism of sialic acids. Cell Mol Life Sci. 1998;54(12):1330–49.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Cheng B, Xie R, Dong L, Chen X. Metabolic remodeling of cell-surface sialic acids: principles, applications, and recent advances. Chembiochem. 2016;17(1):11–27.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Varki A. Sialic acids in human health and disease. Trends Mol Med. 2008;14(8):351–60.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Zhang ZJ, Wuhrer M, Holst S. Serum sialylation changes in cancer. Glycoconj J. 2018;35(2):139–60.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Bardor M, Nguyen DH, Diaz S, Varki A. Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells. J Biol Chem. 2005;280(6):4228–37.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7.

    Rodrigues E, Macauley MS. Hypersialylation in cancer: modulation of inflammation and therapeutic opportunities. Cancers. 2018;10:6.

    Article  CAS  Google Scholar 

  8. 8.

    Zhou XM, Yang GL, Guan F. Biological functions and analytical strategies of sialic acids in tumor. Cells-Basel. 2020;9:2.

    Google Scholar 

  9. 9.

    Pearce OMT, Laubli H. Sialic acids in cancer biology and immunity. Glycobiology. 2016;26(2):111–28.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Wylie AD, Zandberg WF. Quantitation of sialic acids in infant formulas by liquid chromatography-mass spectrometry: an assessment of different protein sources and discovery of new analogues. J Agric Food Chem. 2018;66(30):8114–23.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM. Diversity of microbial sialic acid metabolism. Microbiol Mol Biol R. 2004;68(1):132.

    CAS  Article  Google Scholar 

  12. 12.

    Schauer R. Sialic acids: fascinating sugars in higher animals and man. Zoology. 2004;107(1):49–64.

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Altman MO, Gagneux P. Absence of Neu5Gc and presence of anti-Neu5Gc antibodies in humans-an evolutionary perspective. Front Immunol. 2019;10.

  14. 14.

    Hedlund M, Padler-Karavani V, Varki NM, Varki A. Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression. P Natl Acad Sci USA. 2008;105(48):18936–41.

    CAS  Article  Google Scholar 

  15. 15.

    Samraj AN, Pearce OMT, Laubli H, Crittenden AN, Bergfeld AK, Banda K, et al. A red meat-derived glycan promotes inflammation and cancer progression. P Natl Acad Sci USA. 2015;112(2):542–7.

    CAS  Article  Google Scholar 

  16. 16.

    Padler-Karavani V, Hurtado-Ziola N, Pu MY, Yu H, Huang SS, Muthana S, et al. Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer. Cancer Res. 2011;71(9):3352–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Muchmore EA, Diaz S, Varki A. A structural difference between the cell surfaces of humans and the great apes. Am J Phys Anthropol. 1998;107(2):187–98.

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Irie A, Koyama S, Kozutsumi Y, Kawasaki T, Suzuki A. The molecular basis for the absence of N-glycolylneuraminic acid in humans. J Biol Chem. 1998;273(25):15866–71.

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Dhar C, Sasmal A, Varki A. From "serum sickness" to "xenosialitis": past, present, and future significance of the non-human sialic acid Neu5Gc. Front Immunol. 2019;10.

  20. 20.

    Pearce OMT, Laubli H, Verhagen A, Secrest P, Zhang JQ, Varki NM, et al. Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies. P Natl Acad Sci USA. 2014;111(16):5998–6003.

    CAS  Article  Google Scholar 

  21. 21.

    Pham T, Gregg CJ, Karp F, Chow R, Padler-Karavani V, Cao HZ, et al. Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium. Blood. 2009;114(25):5225–35.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Samraj AN, Laubli H, Varki N, Varki A. Involvement of a non-human sialic acid in human cancer. Front Oncol. 2014;4:33.

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Shewell LK, Wang JJ, Paton JC, Paton AW, Day CJ, Jennings MP. Detection of N-glycolylneuraminic acid biomarkers in sera from patients with ovarian cancer using an engineered N-glycolylneuraminic acid-specific lectin SubB2M. Biochem Biophys Res Commun. 2018;507(1-4):173–7.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Higashi H, Hirabayashi Y, Fukui Y, Naiki M, Matsumoto M, Ueda S, et al. Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer. Cancer Res. 1985;45(8):3796–802.

    CAS  PubMed  Google Scholar 

  25. 25.

    Kawai T, Kato A, Higashi H, Kato S, Naiki M. Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as a tumor-associated sialic acid by gas chromatography-mass spectrometry. Cancer Res. 1991;51(4):1242–6.

    CAS  PubMed  Google Scholar 

  26. 26.

    Koda T, Aosasa M, Asaoka H, Nakaba H, Matsuda H. Application of tyramide signal amplification for detection of N-glycolylneuraminic acid in human hepatocellular carcinoma. Int J Clin Oncol. 2003;8(5):317–21.

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Diaz SL, Padler-Karavani V, Ghaderi D, Hurtado-Ziola N, Yu H, Chen X, et al. Sensitive and specific detection of the non-human sialic acid n-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS One. 2009;4:1.

    Article  CAS  Google Scholar 

  28. 28.

    Nguyen DH, Tangvoranuntakul P, Varki A. Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells. J Immunol. 2005;175(1):228–36.

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Padler-Karavani V, Yu H, Cao HZ, Chokhawala H, Karp F, Varki N, et al. Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease. Glycobiology. 2008;18(10):818–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Rousse J, Salama A, Leviatan Ben-Arye S, Hruba P, Slatinska J, Evanno G, et al. Quantitative and qualitative changes in anti-Neu5Gc antibody response following rabbit anti-thymocyte IgG induction in kidney allograft recipients. Eur J Clin Investig. 2019;49(4):e13069.

    CAS  Article  Google Scholar 

  31. 31.

    Padler-Karavani V, Tremoulet A, Yu H, Chen X, Burns J, Varki A. A simple method for assessment of human anti-Neu5Gc antibodies applied to Kawasaki disease. J Immunol. 2013;190.

  32. 32.

    Tangvoranuntakul P, Gagneux P, Diaz S, Bardor M, Varki N, Varki A, et al. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. P Natl Acad Sci USA. 2003;100(21):12045–50.

    CAS  Article  Google Scholar 

  33. 33.

    Gebrehiwot AG, Melka DS, Kassaye YM, Rehan IF, Rangappa S, Hinou H, et al. Healthy human serum N-glycan profiling reveals the influence of ethnic variation on the identified cancer-relevant glycan biomarkers. PLoS One. 2018;13:12.

    Article  Google Scholar 

  34. 34.

    Hammad LA, Derryberry DZ, Jmeian YR, Mechref Y. Quantification of monosaccharides through multiple-reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column. Rapid Commun Mass Sp. 2010;24(11):1565–74.

    CAS  Article  Google Scholar 

  35. 35.

    Chemmalil L, Suravajjala S, See K, Jordan E, Furtado M, Sun C, et al. A novel approach for quantitation of nonderivatized sialic acid in protein therapeutics using hydrophilic interaction chromatographic separation and nano quantity analyte detection. J Pharm Sci-Us. 2015;104(1):15–24.

    CAS  Article  Google Scholar 

  36. 36.

    Wang LB, Wang D, Zhou X, Wu LJ, Sun XL. Systematic investigation of quinoxaline derivatization of sialic acids and their quantitation applicability using high performance liquid chromatography. RSC Adv. 2014;4(86):45797–803.

    CAS  Article  Google Scholar 

  37. 37.

    Malykh YN, Schauer R, Shaw L. N-glycolylneuraminic acid in human tumours. Biochimie. 2001;83(7):623–34.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Sillanaukee P, Ponnio M, Jaaskelainen IP. Occurrence of sialic acids in healthy humans and different disorders. Eur J Clin Investig. 1999;29(5):413–25.

    CAS  Article  Google Scholar 

  39. 39.

    Haq M, Haq S, Tutt P, Crook M. Serum total sialic-acid and lipid-associated sialic-acid in normal individuals and patients with myocardial-infarction, and their relationship to acute-phase proteins. Ann Clin Biochem. 1993;30:383–6.

    PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Seo Y, Oh MJ, Park JY, Ko JK, Kim JY, An HJ. Comprehensive characterization of biotherapeutics by selective capturing of highly acidic glycans using stepwise PGC-SPE and LC/MS/MS. Anal Chem. 2019;91(9):6064–71.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    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.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Aldredge D, An HJ, Tang N, Waddell K, Lebrilla CB. Annotation of a serum N-glycan library for rapid identification of structures. J Proteome Res. 2012;11(3):1958–68.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Song T, Aldredge D, Lebrilla CB. A method for in-depth structural annotation of human serum glycans that yields biological variations. Anal Chem. 2015;87(15):7754–62.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Kim U, Oh MJ, Seo Y, Jeon Y, Eom JH, An HJ. Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein. Bioanalysis. 2017;9(18):1373–83.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    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):4414–23.

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Hua S, Jeong HN, Dimapasoc LM, Kang I, Han C, Choi JS, et al. Isomer-specific LC/MS and LC/MS/MS profiling of the mouse serum N-glycome revealing a number of novel sialylated N-glycans. Anal Chem. 2013;85(9):4636–43.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Chao Q, Ding Y, Chen ZH, Xiang MH, Wang N, Gao XD. Recent progress in chemo-enzymatic methods for the synthesis of N-glycans. Front Chem. 2020;8.

  48. 48.

    Ashwood C, Lin CH, Thaysen-Andersen M, Packer NH. Discrimination of isomers of released N- and O-glycans using diagnostic product ions in negative ion PGC-LC-ESI-MS/MS. J Am Soc Mass Spectrom. 2018;29(6):1194–209.

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Ruhaak LR, Lebrilla CB. Applications of multiple reaction monitoring to clinical glycomics. Chromatographia. 2015;78(5-6):335–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Kumozaki S, Sato K, Sakakibara Y. A machine learning based approach to de novo sequencing of glycans from tandem mass spectrometry spectrum. Ieee Acm T Comput Bi. 2015;12(6):1267–74.

    CAS  Google Scholar 

  51. 51.

    Lowenthal MS, Kilpatrick EL, Phinney KW. Separation of monosaccharides hydrolyzed from glycoproteins without the need for derivatization. Anal Bioanal Chem. 2015;407(18):5453–62.

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    Hammad LA, Saleh MM, Novotny MV, Mechref Y. Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins. J Am Soc Mass Spectrom. 2009;20(6):1224–34.

    CAS  PubMed  Article  Google Scholar 

  53. 53.

    Lee HHL, Heo CE, Seo N, Yun SG, An HJ, Kim HI. Accurate quantification of N-glycolylneuraminic acid in therapeutic proteins using supramolecular mass spectrometry. J Am Chem Soc. 2018;140(48):16528–34.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Xu GG, Amicucci MJ, Cheng Z, Galermo AG, Lebrilla CB. Revisiting monosaccharide analysis - quantitation of a comprehensive set of monosaccharides using dynamic multiple reaction monitoring. Analyst. 2018;143(1):200–7.

    CAS  Article  Google Scholar 

  55. 55.

    van der Ham M, Prinsen BH, Huijmans JG, Abeling NG, Dorland B, Berger R, et al. Quantification of free and total sialic acid excretion by LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci. 2007;848(2):251–7.

    Article  CAS  Google Scholar 

  56. 56.

    Ji SN, Wang F, Chen Y, Yang CW, Zhang PW, Zhang XB, et al. Developmental changes in the level of free and conjugated sialic acids, Neu5Ac, Neu5Gc and KDN in different organs of pig: a LC-MS/MS quantitative analyses. Glycoconj J. 2017;34(1):21–30.

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Veillon L, Huang YF, Peng WJ, Dong X, Cho BG, Mechref Y. Characterization of isomeric glycan structures by LC-MS/MS. Electrophoresis. 2017;38(17):2100–14.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. 58.

    Ruhaak LR, Zauner G, Huhn C, Bruggink C, Deelder AM, Wuhrer M. Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem. 2010;397(8):3457–81.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Packer NH, Lawson MA, Jardine DR, Redmond JW. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj J. 1998;15(8):737–47.

    CAS  PubMed  Article  Google Scholar 

  60. 60.

    Monser L. Liquid chromatographic determination of four purine bases using porous graphitic carbon column. Chromatographia. 2004;59(7-8):455–9.

    CAS  Article  Google Scholar 

  61. 61.

    Tornkvist A, Markides KE, Nyholm L. Chromatographic behaviour of oxidised porous graphitic carbon columns. Analyst. 2003;128(7):844–8.

    Article  Google Scholar 

  62. 62.

    Jensen PH, Karlsson NG, Kolarich D, Packer NH. Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc. 2012;7(7):1299–310.

    CAS  PubMed  Article  Google Scholar 

  63. 63.

    Ashwood C, Pratt B, MacLean BX, Gundry RL, Packer NH. Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping. Analyst. 2019;144(11):3601–12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. 64.

    Chang R, Yang B, Zhu QJ. Theoretical studies on the electronic structure parameters and reactive activity of Neu5Gc and Neu5Ac under food processing solvent environment. Molecules. 2019;24:2.

    Google Scholar 

  65. 65.

    McCalley DV. Understanding and manipulating the separation in hydrophilic interaction liquid chromatography. J Chromatogr A. 2017;1523:49–71.

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Angeles LF, Aga DS. Establishing analytical performance criteria for the global reconnaissance of antibiotics and other pharmaceutical residues in the aquatic environment using liquid chromatography-tandem mass spectrometry. J Anal Methods Chem. 2018.

  67. 67.

    Clerc F, Reiding KR, Jansen BC, Kammeijer GS, Bondt A, Wuhrer M. Human plasma protein N-glycosylation. Glycoconj J. 2016;33(3):309–43.

    CAS  PubMed  Article  Google Scholar 

  68. 68.

    Kronewitter SR, de Leoz MLA, Peacock KS, McBride KR, An HJ, Miyamoto S, et al. Human serum processing and analysis methods for rapid and reproducible N-glycan mass profiling. J Proteome Res. 2010;9(10):4952–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references


This research was supported by a grant (21172MFDS192) from the Ministry of Food and Drug Safety in 2021.

Author information




Conceptualization, Y.J. Choi and H.J. An. Methodology, N. Seo, J. Ko, and H.J. An. Validation, N. Seo, U. Kim, M.J. Oh, and H.J. An. Investigation, N. Seo, J. Ko, and D. Lee. Formal analysis, N. Seo, J. Ko, D. Lee, and H. Jeong. Resources, Y.J. Choi, D.H. Lee, J. Kim, and H.J. An. Writing original draft preparation, N. Seo and H.J. An. Writing review and editing, N. Seo and H.J. An. Supervision, Y.J. Choi and H.J. An. Funding acquisition, H.J. An.

Corresponding authors

Correspondence to Yoon Jin Choi or Hyun Joo An.

Ethics declarations

Ethics approval and consent to participate

Human sera were collected by the Seoul National University Bundang Hospital (SNUH) by means of a standardized protocol approved by the SNUH Institutional Review Board (B-1607/353-001). Informed consent was provided by all individuals involved in the study.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information


(DOCX 829 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Seo, N., Ko, J., Lee, D. et al. In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS. Anal Bioanal Chem 413, 5227–5237 (2021).

Download citation


  • Sialic acid
  • Neu5Gc
  • Absolute quantitation
  • Human serum
  • Label-free MRM-MS