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 (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) 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 TiO(2) resin. By combining TiO(2) 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.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N- The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs) 1 is an attractive tool for researchers. There is increasing evidence that both phosphorylation and glycosylation play important roles in cellular signaling networks during development and transformation of cells. Development of the mammalian brain is initiated during the embryonic stage and continues until adulthood. The brain originates through the proliferation of the telencephalon, the anterior part of the neural tube. Following differentiation, cells begin to migrate and associate into different brain structures. The brain structures are reorganized with the extension of axons and dendrites to communicate via synaptic terminal interactions (1, 2). These molecular interactions are governed by cell surface receptors that are often post-translationally modified with both N-linked glycans and phosphate groups, and studies have suggested that extracellular glycans play vital roles in the regulation of signal transduction pathways (3). For example, the myelin-associated glycoprotein (MAG) binds to cell surface glyco-conjugates GD1a, GT1b and Nogo receptors to form signaling complexes that inhibit axon outgrowth, whereas inhibition of Rho kinase reverses this process in a number of nerve cell types (4). There is growing evidence that both the differentiation and migration of neurons and the guidance of axons are regulated by sialic acid-containing glycoconjugates (5-7). 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-

Glycosylation is one of the many post-translational protein modifications that regulate several biological processes of proteins and lipids. In particular aberrant sialylation, at the terminal position of the glycan structures of cell surface proteins, occurs in numerous diseases such as cancer metastasis and viral infections. Methodological improvements in the sample preparation and analysis currently enable the detailed identification of the glycosylation sites and glycan structure characterization. In this context, the aim of this chapter is to describe a methodology to identify the glycosylation site of N-linked sialylated glycoproteins. The method relies on the specificity of titanium dioxide affinity chromatography to isolate sialic acid-containing glycopeptides. After enzymatic release of the glycans, the enriched sialylated glycopeptides are analyzed by mass spectrometry. This strategy was applied to a crude membrane fraction of EGF-stimulated HeLa cells metabolically labeled with SILAC enabling both qualitative and quantitative analyses of sialoglycopeptides.

In recent years, production of biosynthesized human milk oligosaccharides (HMOs) has become scalable to industrial standards. As a result, infant formula fortified with 2'-fucosyllactose (2'-FL), the most abundant HMO in human breast milk, is now commercially available. 2'-FL and 3-fucosyllactose (3-FL), another abundant HMO, are thought to be beneficial for infant health and development. Products containing HMOs are projected to expand in the future, showing the need for robust, easily applicable analytical methods for the quantitative assessment of HMOs in different food applications. We present here a validated high-performance liquid chromatography method for the quantification of 2'-FL and 3-FL in whole milk, infant formula, and cereal bars. The sample preparation was simple dispersion and extraction of the sample. The samples were analyzed by hydrophilic interaction liquid chromatography with refractive index detection and a runtime of 19 min. The method had a high degree of linearity (R 2 > 0.9995) in the range 0.2 to 12 mg/mL. The recovery for 2'-FL was 88% to 105% and for 3-FL 94% to 112%. The limit of detection (LOD) for whole milk was 0.1 mg/mL for 2'-FL and 0.2 mg/mL for 3-FL. In infant formula and cereal bars, the LOD was 0.6 mg/g for both 2'-FL and 3-FL. To show the practical application of this method, it was successfully utilized in stability studies of 2'-FL and 3-FL in whole milk, UHT milk, and yoghurt. The method provides a means of simultaneous and robust quantification of 2'-FL and 3-FL in various food matrices with high accuracy and high reproducibility.Practical Application: 2'-Fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are two of the most abundant human milk oligosaccharides (HMOs) present in human breast milk. We present a fast HPLC method for the robust quantification of these two compounds in infant formula, whole milk, UHT milk, cereal bars, and yoghurt. This method can easily be set up by food producers and researchers to analyze the dosage of 2'-FL and 3-FL in their product or perform shelf life studies in different food applications.