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Special Issue: 8th International Symposium On Mass Spectrometry In The Life Sciences

Quantitative Analysis of Synaptic Phosphorylation and Protein Expression^*,S

Jonathan C. Trinidad, Agnes Thalhammer, Christian G. Specht^,¶, Aenoch J. Lynn, Peter R. Baker, Ralf Schoepfer^,|| and Alma L. Burlingame^,**

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

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength.

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