Analytical and Bioanalytical Chemistry

, Volume 409, Issue 21, pp 4971–4981 | Cite as

Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples

  • Na Young Choi
  • Heeyoun Hwang
  • Eun Sun Ji
  • Gun Wook Park
  • Ju Yeon Lee
  • Hyun Kyoung Lee
  • Jin Young KimEmail author
  • Jong Shin YooEmail author
Research Paper


Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell–cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 μg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.


Dried blood spot N-glycosylation Glycoproteomics 



This research was supported by the National Research Council of Science and Technology (NTM2371511, the Creative Allied Project (CAP)), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI); it was funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C2098) and by Korea Basic Science Institute grant (T37413, P.I. KJY).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethics approval and consent to participate

This study was performed in accordance with ethical standards. Blood sample was obtained from voluntary blood donors with informed consent and approval in accordance with IRB guidelines from Yonsei University College of Medicine (Seoul, Korea).

Supplementary material

216_2017_438_MOESM1_ESM.pdf (470 kb)
ESM 1 (PDF 470 kb)
216_2017_438_MOESM2_ESM.xlsx (2.1 mb)
ESM 2 (XLSX 2190 kb)


  1. 1.
    Chace DH, Kalas TA, Naylor EW. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem. 2012;49:1797–817.CrossRefGoogle Scholar
  2. 2.
    Sahai I, Marsden D. Newborn screening. Crit Rev Clin Lab Sci. 2009;46:55–82.CrossRefGoogle Scholar
  3. 3.
    Plamen AD. Dried blood spots: analysis and applications. Anal Chem. 2013;85:779–89.CrossRefGoogle Scholar
  4. 4.
    Chambers AG, Percy AJ, Yang J, Camenzind AG, Borchers CH. Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoring–mass spectrometry. Mol Cell Proteomics. 2013;12:781–91.CrossRefGoogle Scholar
  5. 5.
    Chambers AG, Percy AJ, Hardie DB, Borchers CH. Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS. J Am Soc Mass Spectrom. 2013;24:1338–45.CrossRefGoogle Scholar
  6. 6.
    Chamber AG, Percy AJ, Yang J, Bochers CH. Multiple reaction monitoring enables precise quantification of 97 proteins in dried blood spots. Mol Cell Proteomics. 2015;14(11):3094–104.CrossRefGoogle Scholar
  7. 7.
    Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, et al. Essentials of glycobiology. 2nd ed. New York: Cold Spring Harbor; 2009.Google Scholar
  8. 8.
    Wormald MR, Dwek RA. Glycoproteins: glycan presentation and protein-fold stability. Structure. 1999;7:155–60.CrossRefGoogle Scholar
  9. 9.
    Quast I, Peschke B, Lünemann JD. Regulation of antibody effector functions through IgG Fc N-glycosylation. Cell Mol Life Sci. 2017;74(5):837–47.CrossRefGoogle Scholar
  10. 10.
    Reiding KR, Ruhaak LR, Uh HW, El Bouhaddani S, van den Akker EB, Plomp R, et al. Wuhrer human plasma N-glycosylation as analyzed by matrix-assisted laser desorption/ionization-fourier transform ion cyclotron resonance-MS associates with markers of inflammation and metabolic health. Mol Cell Proteomics. 2016;16:228–42.CrossRefGoogle Scholar
  11. 11.
    Balog CI, Stavenhagen K, Fung WL, Koeleman CA, McDonnell LA, Verhoeven A, et al. N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation. Mol Cell Proteomics. 2012;11(9):571–85.CrossRefGoogle Scholar
  12. 12.
    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
  13. 13.
    Kornfeld R, Koenfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–64.CrossRefGoogle Scholar
  14. 14.
    Ruhaak LR, Miyamoto S, Kelly K, Lebrilla CB. N-glycan profiling of dired blood spot. Anal Chem. 2012;84:396–402.CrossRefGoogle Scholar
  15. 15.
    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 Biophy Acta. 1844;2014:1437–52.Google Scholar
  16. 16.
    Ahn YH, Kim JY, Yoo JS. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods. Mass Spec Rev. 2015;34:148–65.CrossRefGoogle Scholar
  17. 17.
    Hwang H, Lee JY, Lee HK, Park GW, Jeong HK, Moon MH, et al. In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. Anal Bioanal Chem. 2014;406:7999–8011.CrossRefGoogle Scholar
  18. 18.
    Kim KH, Ahn YH, Ji ES, Lee JY, Kim JY, An HJ, et al. Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry. Anal Chim Acta. 2015;882:38–48.CrossRefGoogle Scholar
  19. 19.
    Lee JY, Lee HK, Park GW, Hwang H, Jeong HK, Yun KN, et al. Characterization of site-specific N-glycopeptide isoforms of α-1-acid glycoprotein from an interlaboratory study using LC-MS/MS. J Proteome Res. 2016;15(12):4146–64.CrossRefGoogle Scholar
  20. 20.
    Ji ES, Hwang H, Park GW, Lee JY, Lee HK, Choi NY, et al. Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry. Anal Bioanal Chem. 2016;408(27):7761–74.CrossRefGoogle Scholar
  21. 21.
    Park GW, Kim JY, Hwang H, Lee JY, Ahn YH, Lee HK, et al. Integrated GlycoProteome Analyzer (I-GPA) for automated identification and quantification of site-specific N-glycosylation. Sci Rep. 2016;6:21175.CrossRefGoogle Scholar
  22. 22.
    Segu ZM, Hammad LA, Mechref Y. Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion. Rapid Comm Mass Spec. 2010;24(23):3461–8.CrossRefGoogle Scholar
  23. 23.
    An HJ, John WF, Carlito BL. Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol. 2009;13:421–6.CrossRefGoogle Scholar
  24. 24.
    Gordon JA, Jencks WP. The relationship of structure to the effectiveness of denaturing agents for proteins. Biochemist. 1963;2:47–57.CrossRefGoogle Scholar
  25. 25.
    Rajagopalan KV, Fridovich I, Handler P. Competitive inhibition of enzyme activity by urea. J Biol Chem. 1961;236:1059–65.Google Scholar
  26. 26.
    Kakhniashvili DG, Bulla LA, Goodman SR. The human erythrocyte proteome. Mol Cell Proteomics. 2004;3:501–9.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Na Young Choi
    • 1
    • 2
  • Heeyoun Hwang
    • 1
  • Eun Sun Ji
    • 1
  • Gun Wook Park
    • 1
  • Ju Yeon Lee
    • 1
  • Hyun Kyoung Lee
    • 1
    • 2
  • Jin Young Kim
    • 1
    Email author
  • Jong Shin Yoo
    • 1
    • 2
    Email author
  1. 1.Biomedical Omics GroupKorea Basic Science InstituteCheongjuRepublic of Korea
  2. 2.Graduated School of Analytical Science and TechnologyChungnam National UniversityDaejeonRepublic of Korea

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