Selective Enrichment of Sialylated Glycopeptides Using TiO2 Beads

Affinity purification for mass spectrometry based on titanium dioxide beads

by Julian Saba, Ph.D.1; Rosa Viner, Ph.D.1 - 06/01/11

Sialylated glycans and glycoproteins are implicated in numerous physiopathological mechanisms and used in applications for biotherapeutic development. Mass spectrometry (MS) has become one of the most powerful tools for glycopeptide analysis; however, structural characterization of sialylated glycopeptides remains analytically challenging because of their low abundance, and acidic and hydrophilic properties. In principle, targeted enrichment of sialylated glycopeptides would ensure utilization of MS to its full unique ability for extracting all potentially accessible information from a sample. Enrichment would greatly reduce complexity of the overall sample matrix, thereby enabling more sensitive and accurate analysis. The Thermo Scientific Pierce TiO₂ Phosphopeptide Enrichment and Clean-up Kit enables efficient isolation of sialylated glycopeptides from complex and fractionated protein digests for analysis by MS.

Alterations in sialylation are linked to development, tumor biology and autoantigenicity, and other diseases, and sialylation has been shown to significantly increase biopharmaceutical half-life and activity in vivo.¹ The high selectivity of TiO₂ toward negatively charged sialylated glycopeptides results in highly specific binding of these species. This phosphopeptide enrichment and clean-up kit contains TiO₂ spin tips, graphite spin columns, buffers and an easy-to- use protocol that produces high yield of phosphopeptides and clean glycopeptide samples ready for MS analysis. The kit is compatible with our lysis, reduction, alkylation and digestion reagents to provide a complete workflow for studying sialylated glycopeptides.

Comprehensive glycosylation-site profiling of a single glycoprotein

Efficient enrichment of sialylated N-linked glycopeptides using TiO₂ has been reported.^2,3 Graphite columns have affinity for glycopeptides with a smaller peptide backbone.⁴ To demonstrate the utility and specificity of TiO₂ for enriching sialylated glycopeptides, TiO₂ and graphite were used alone or combined for a two-step enrichment. This approach ensures all sizes of glycopeptides are enriched for analysis and compared against the most commonly used zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC)-based strategy. Bovine alpha-1-acid glycoprotein (AGP) was used as the model system for our method development because of its high glycan content, multiple glycosylation sites, and the presence of small glycopeptides. Additionally, this protein is well characterized in terms of glycosylation patterns, and extensive reference data are readily available in the literature.⁵ Comparison of the efficiency of various enrichment strategies was examined (Figure 1). Bovine AGP contains five N-glycosylation sites containing complex-type glycan structures, with a significant portion being highly sialylated multi-antennary. Of all the tested enrichment approaches, only TiO₂ alone or in combination with Tandem Mass Tag* (TMT*) Reagents and graphite columns enabled detection and identification of all five N-glycosylation sites and their multiple glycopeptide glycoforms.

Figure 1. Comparison of ZIC-HILIC vs. TiO2 vs. TMT-TiO2-graphite vs. graphite for enrichment of bovine AGP glycopeptides.

Comprehensive sialyl glycosylation-site profiling of human serum glycoproteome

To test the performance of TiO₂ with complex samples, a human serum tryptic digest treated with H2¹⁸O-PGN Fase was examined. | Results from the two enrichment strategies (TiO₂ vs. ZIC-HILIC) for human serum are summarized in Figure 2. Both approaches had high numbers of unique glycopeptides and glycoproteins with overlap of only 35%. Our results for neat glycopeptides corroborate the observation that the glycopeptides present in the Venn diagram for TiO₂ were N-linked and acidic, while unique glycopeptides in ZIC-HILIC were mainly N-linked and neutral.

Figure 2. Comparison of TiO2 vs ZIC-HILIC for enrichment of human serum glycopeptides.

Examples of identified sialylated N-linked glycopeptides unique to TiO₂-based enrichment strategy are shown in Figure 3. The sialylated glycopeptides of histidine-rich glycoprotein were identified only in the TiO₂-based enrichment strategy. Overall, TiO₂ and ZIC-HILIC provided complementary enrichment strategies, and TiO₂ selectively enriched sialylated glycopeptides.

1. Essentials of Glycobiology, 2nd edition (2009). Edited by Varki, A. et al. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. pp 199-218, 622-623, 722-724.
2. Takegawa, Y., et al. (2006). Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography. J Sep Sci 16:2533-40.
3. Larsen, M.R., et al. (2007). Exploring the sialiome using titanium dioxide chromatography and mass spectrometry. Mol Cell Proteomics 6:1778-87.
4. Larsen, M.R., et al. (2005). Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry. Mol Cell Proteomics 4:107-19.
5. Treuheit, M.J., et al. (1992). Analysis of the five glycosylation sites of human alpha 1-acid glycoprotein. Biochem J 283:105-12.