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

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Mass Spectrometry of Glycoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 951))

Abstract

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.

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Acknowledgments

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

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Authors and Affiliations

  1. Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Pia Hønnerup Jensen

  2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Simon Mysling, Peter Højrup & Ole Nørregaard Jensen

Authors
  1. Pia Hønnerup Jensen
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  2. Simon Mysling
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  3. Peter Højrup
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  4. Ole Nørregaard Jensen
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Corresponding author

Correspondence to Pia Hønnerup Jensen .

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Editors and Affiliations

  1. Medical Center, Department of Biochemistry, University of Texas Southwestern, Harry Hines Blvd. 5323, Dallas, 75390-9038, Texas, USA

    Jennifer J. Kohler

  2. Medical Center, Dallas, University of Texas Southwestern, Harry Hines Blvd. 5323, Dallas, 75390-9185, Texas, USA

    Steven M. Patrie

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Jensen, P.H., Mysling, S., Højrup, P., Jensen, O.N. (2013). Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE). In: Kohler, J., Patrie, S. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 951. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-146-2_10

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  • DOI: https://doi.org/10.1007/978-1-62703-146-2_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-145-5

  • Online ISBN: 978-1-62703-146-2

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