Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)
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 wordsGlycopeptides 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.”
- 3.Varki A, Lowe JB (1999) Biological roles of glycans. In: Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J (eds) Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Woodbury, NY, pp 57–68Google Scholar
- 9.Kieliszewski MJ, O’Neill M, Leykam J, Orlando R (1995) Tandem mass spectrometry and structural elucidation of glycopeptides from a hydroxyproline-rich plant cell wall glycoprotein indicate that contiguous hydroxyproline residues are the major sites of hydroxyproline O-arabinosylation. J Biol Chem 270:2541–2549CrossRefPubMedGoogle Scholar
- 24.Højrup P (2009) Peptide mapping for protein characterization. In: Walker JM (ed) The protein protocols handbook, 3rd edn. Humana Press, Totawa, NJ, pp 965–983Google Scholar