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.
Highlights
From 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; ʈSchool of Molecular Bioscience, University of Sydney, New South Wales, 2006, Australia; **Biomedical Laboratory, Odense University Hospital, University of Southern Denmark, 5000 Odense C, Denmark
The phosphopeptides and formerly N-linked sialylated glycopeptides were separated by hydrophilic interaction liquid chromatography (HILIC) into 14 fractions (Fig. 1B) prior to mass spectrometry (MS)/MS
The utility of HPLC HILIC enables the separation of formerly sialylated glycopeptides and phosphopeptides after an optimized TiO2 enrichment
Summary
From 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; ʈSchool of Molecular Bioscience, University of Sydney, New South Wales, 2006, Australia; **Biomedical Laboratory, Odense University Hospital, University of Southern Denmark, 5000 Odense C, Denmark. Linked sialylated glycopeptides and phosphopeptides from complex membrane protein preparations of a variety of biological samples. The eluted peptides were dried by vacuum centrifugation to produce the enriched phosphopeptide/ sialylated glycopeptide fraction.
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