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Phosphoproteomic Analysis of Synaptosomes from Human Cerebral Cortex

Joseph A. DeGiorgis,^† Howard Jaffe,^‡ Jorge E. Moreira,^§ Carlos G. CarlottiJr., João P. Leite,^# Harish C. Pant, and Ayse Dosemeci*^†

Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Protein and Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Department of Cellular and Molecular Biology, Ribeiro Preto School of Medicine, University of So Paulo, Ribeiro Preto, SP, Brasil, Division of Neurosurgery, Department of Surgery, Ribeiro Preto School of Medicine, University of So Paulo, Ribeiro Preto, SP, Brasil, Department of Neurology, Ribeiro Preto School of Medicine, University of So Paulo, Ribeiro Preto, SP, Brasil, Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543

J. Proteome Res., 2005, 4 (2), pp 306–315

DOI: 10.1021/pr0498436

Publication Date (Web): February 5, 2005

Marine Biological Laboratory.

†

Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health.

‡

Protein and Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health.

Department of Cellular and Molecular Biology, Ribeirão Preto School of Medicine, University of São Paulo.

Division of Neurosurgery, Department of Surgery, Ribeirão Preto School of Medicine, University of São Paulo.

Department of Neurology, Ribeirão Preto School of Medicine, University of São Paulo.

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health.

To whom correspondence should be addressed. 9000 Rockville Pike, NIH Bldg 36, 2A21, Bethesda, MD 20892. Phone: (301) 435-2795. Fax: (301) 480-1485. E-mail: dosemeca@mail.nih.gov.

Abstract

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.

Keywords: LC−MS/MS • IMAC • mass spectrometry • protein phosphorylation • phosphoproteomics • synaptosome • synaptic • synataxin • SNIP • GABA-B

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