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Originally published In Press as doi:10.1074/mcp.T500041-MCP200 on February 1, 2006.
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Molecular & Cellular Proteomics 5:914-922, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Comprehensive Identification of Phosphorylation Sites in Postsynaptic Density Preparations*,S

Jonathan C. Trinidad{ddagger}, Christian G. Specht§,¶, Agnes Thalhammer§, Ralf Schoepfer§,|| and Alma L. Burlingame{ddagger},**

From the {ddagger} Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 § Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and ~80% of identified phosphorylation sites are novel.

** To whom correspondence may be addressed. E-mail: alb{at}cgl.ucsf.edu


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