Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes
Experimental & Molecular Medicine
; : e29-2013.
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| ID: wpr-74491
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WPRO
ABSTRACT
O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.
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Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Péptidos
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Fosforilación
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Acetilglucosamina
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Sinaptosomas
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Glicosilación
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Encéfalo
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Datos de Secuencia Molecular
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Cromatografía de Afinidad
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Secuencia de Aminoácidos
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Sinapsinas
Límite:
Animals
Idioma:
En
Revista:
Experimental & Molecular Medicine
Año:
2013
Tipo del documento:
Article