Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry

Giuseppe Palmisano, Sara Eun Lendal, Kasper Engholm-Keller, Rikke Leth-Larsen, Benjamin L. Parker, Martin R. Larsen

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163 Citations (Scopus)


The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO2) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO2 toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO2 resin. By combining TiO2 enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO 2/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.

Original languageEnglish
Pages (from-to)1974-1982
Number of pages9
JournalNature Protocols
Issue number12
Publication statusPublished - 1 Nov 2010
Externally publishedYes


ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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