title: A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development.
abstract: 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-linked 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.
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, and Biomedical Laboratory, Odense University Hospital, Campusvej 55, 5230 Odense M, Denmark.
authors: Giuseppe
authors: G
authors: Palmisano
authors: Benjamin L
authors: BL
authors: Parker
authors: Kasper
authors: K
authors: Engholm-Keller
authors: Sara Eun
authors: SE
authors: Lendal
authors: Katarzyna
authors: K
authors: Kulej
authors: Melanie
authors: M
authors: Schulz
authors: Veit
authors: V
authors: Schwämmle
authors: Mark E
authors: ME
authors: Graham
authors: Henrik
authors: H
authors: Saxtorph
authors: Stuart J
authors: SJ
authors: Cordwell
authors: Martin R
authors: MR
authors: Larsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000254:growth & development* / Q000378:metabolism*; D016000:Cluster Analysis; D017143:Fuzzy Logic; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
chemical_list: D006020:Glycopeptides; C108768:Ncam2 protein, mouse; D019006:Neural Cell Adhesion Molecules; D010748:Phosphopeptides; D019158:N-Acetylneuraminic Acid