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

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 Nlinked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017509, 1191–1202, 2012. The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs) 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 surFrom the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark; §Discipline of Pathology, School of Medical Sciences, University of Sydney, New South Wales, 2006, Australia; ¶Cell Signalling Unit, Children’s Medical Research Institute, University of Sydney, New South Wales, 2145, Australia; School of Molecular Bioscience, University of Sydney, New South Wales, 2006, Australia; **Biomedical Laboratory, Odense University Hospital, University of Southern Denmark, 5000 Odense C, Denmark Received January 26, 2012, and in revised form, July 9, 2012 Published, MCP Papers in Press, July 26, 2012, DOI 10.1074/ mcp.M112.017509 1 The abbreviations used are: PTM, post-translational modification; MAG, myrlin associated glycoprotein; TiO2, titanium dioxide; HILIC, hydrophilic interaction liquid chromatography; TEAB, triethylammonium bicarbonate; iTRAQ, isobaric tracking for relative quantification; ADAM, A disintegrin and metalloproteases; NCAM, neural cell adhesion molecule. Research