Quantitative Analysis of Phosphoproteins in a Parkinson’s Disease Model using Phosphoprotein Isotope-coded Solid-phase Tags
David Cawthon1, Erik J. Soderblom2, Z. Alex Xu1, Helen Duhart1, William Slikker Jr.1, Syed Ali1, and
Michael B. Goshe2
1
Neurochemistry Laboratory, Division of Neurotoxicology, NCTR/FDA, 3900 NCTR Road, Jefferson, AR 72079
2
Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh NC 27695-7622
Received 21 December 2004; received in revised form 3 February 2005; accepted 4 February 2005
Correspondence and requests for reprints should be addressed to:
Dr. David Cawthon
Neurochemistry Laboratory
Division of Neurotoxicology
National Center for Toxicological Research
3900 NCTR Road
Jefferson, AR 72075
Phone: 870-543-7082
Fax: 870-543-7745
email: david.cawthon@fda.hhs.gov
Dr. Michael B. Goshe
Department of Molecular and Structural Biochemistry
North Carolina State University
128 Polk Hall
Campus Box 7622
Raleigh NC 27695-7622
Phone: 919-513-7740
Fax: 919-515-2047
email: michael_goshe@ncsu.edu
Abstract
Objectives
Protein phosphorylation was examined in an in vitro model of Parkinson’s disease during exposure to the neurotoxicant N-methyl-4-phenylpyridinium (MPP+), a metabolite of 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP).
Methodology
A murine dopaminergic cell line (MN9D) was treated with MPTP. The phosphoproteins identified were compared to those of the untreated control using an improved Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) labeling method to isolate and measure the relative abundance of serine and threonine phosphorylation.
Results
Utilizing the improved PhIST approach, 63 phosphoproteins were identified by peptides derivatized with either light (12C6,14N) or heavy (13C6,15N) PhIST tags. The detected proteins belong to several functional classes that include zinc-finger proteins, receptor and transporter proteins, apoptosis and ATP-related proteins. Quantitation of the PhIST-labeled peptides indicated that approximately 45% of the phosphoproteins were unique to the MPTP treated sample, including many that were previously unknown.
Conclusion
The application of PhIST analysis using an in vitro model provides new insights and promising research avenues into the pathophysiology of Parkinson’s disease and demonstrates its utility as a discovery tool for identifying potentially important phosphorylation events.
Key Words
Phosphorylation, Parkinson’s disease, mass spectrometry, stable isotope labeling, liquid chromatography
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