Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)

  • Pia Hønnerup Jensen
  • Simon Mysling
  • Peter Højrup
  • Ole Nørregaard Jensen
Part of the Methods in Molecular Biology book series (MIMB, volume 951)


Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifluoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.

Key words

Glycopeptides Enrichment HILIC MALDI-TOF MS SPE N-glycosylation Ion-pairing reagent 



PHJ was supported by a postdoctoral fellowship from “Annie og Otto Johs. Detlefs’ Almennyttige Fond.”


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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Pia Hønnerup Jensen
    • 1
  • Simon Mysling
    • 2
  • Peter Højrup
    • 2
  • Ole Nørregaard Jensen
    • 2
  1. 1.Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

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