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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

Abstract

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.

Keywords

Dried blood spot N-glycosylation Glycoproteomics 

Notes

Acknowledgments

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)

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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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