Analytical and Bioanalytical Chemistry

, Volume 402, Issue 9, pp 2765–2776 | Cite as

Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

  • Chu-Wei Kuo
  • I-Lin Wu
  • He-Hsuan Hsiao
  • Kay-Hooi KhooEmail author
Original Paper


Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe3O4 nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.


Glycoproteomics N-glycosylation sites Glycopeptide capture Nanoparticles Mass spectrometry 













Hydrophilic interaction liquid chromatography


Mass spectrometry


N-Acetylneuraminic acid


Uterine luminal fluid



This work was supported by Taiwan NSC grants 94-3112-B-001-009-Y and 95-3112-B-001-014 to the NRPGM Core Facilities for Proteomics located at the Institute of Biological Chemistry, Academia Sinica. The LTQ-Orbitrap data were additionally acquired at the Academia Sinica common mass spectrometry facilities, also at the Institute of Biological Chemistry. We are grateful to Dr. Sin-Tak Chu (Institute of Biochemical Sciences, National Taiwan University) for providing the mouse uterine luminal fluid samples.

Supplementary material

216_2012_5724_MOESM1_ESM.pdf (1.7 mb)
ESM 1 (PDF 1.65 mb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Chu-Wei Kuo
    • 1
  • I-Lin Wu
    • 1
  • He-Hsuan Hsiao
    • 1
  • Kay-Hooi Khoo
    • 1
    Email author
  1. 1.NRPGM Core Facilities for Proteomics and Institute of Biological ChemistryAcademia SinicaNankangTaiwan

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