Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry

Abstract

Strategies for biomarker discovery increasingly focus on biofluid protein and peptide expression patterns. Posttranslational modifications contribute significantly to the pattern complexity and thereby increase the likelihood of obtaining specific biomarkers for diagnostics and disease monitoring. Glycosylation is a common post-translational modification that plays a role e.g. in cell adhesion and in cell-cell and receptor-ligand interactions. Abnormal protein glycosylation has important disease associations, and the glycoproteome is therefore a target for biomarker discovery. Here we present a simple and highly selective strategy for purification of sialic acid-containing glycopeptides (the sialiome) from complex peptide mixtures. The approach utilizes a high and selective affinity of sialic acids for titanium dioxide under specific buffer conditions. In combination with mass spectrometry we used this strategy to characterize the human plasma and saliva sialiomes where 192 and 97 glycosylation sites, respectively, were identified. Furthermore we illustrate the potential of this method in biomarker discovery.

A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

“…Dietary supplementation of sialic acid leads to increases in sialic acid-containing glycoproteins in the frontal cortex and is associated with faster learning and memory in piglets (8). The nervous system contains an abundant array of sialylated molecules and it is therefore not sur-prising that changes in the sialiome (the content of sialylated glycoproteins (9)) of a neuron can regulate activity. Removal of sialic acids from membrane proteins by NEU3 in primary neurons leads to actin depolymerization and axonal growth through TrkA-mediated signaling (10).…”

“…However, using this procedure resulted in significant co-enrichment of nonphosphorylated peptides. Later we demonstrated that TiO 2 was able to selectively purify phosphorylated peptides and sialic acid-containing N-glycopeptides (9,17) if peptide samples are loaded onto the TiO 2 resin in a buffer containing high organic solvent, very low pH and a multifunctional acid, such as 2,5-dihydroxybenzoic acid or glycolic acid.…”

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

“…NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZICா-HILIC column (23)(24)(25)(26)(27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g.…”

“…Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23)(24)(25)(26)(27)(28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZICா-HILIC column (23)(24)(25)(26)(27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28).…”

Large‐scale N‐glycoproteome map of rat brain tissue: Simultaneous characterization of insoluble and soluble protein fractions

“…Therefore, high efficient and specific enrichment procedure is imperative for large-scale glycoproteome analysis. Recently, various methods, including hydrophilic interaction liquid chromatography [5,6], lectinbased affinity chromatography [7][8][9], hydrazide chemistry [10] and TiO 2 [11,12], have been developed, and further used for the selective enrichment of glycoproteins/glycopeptides. Among above-mentioned methods, Hydrazide gel-based solid-phase extraction becomes a popular technique, mainly due to its high efficiency and excellent selectivity, and has been used for the analysis of N-glycoproteome in various biological samples [10,[13][14][15].…”