title: Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.
abstract: Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.
authors: †Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States.
authors: Mark S
authors: MS
authors: Lowenthal
authors: †Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States.|‡Laboratory of Neurotoxicology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors: Sanford P
authors: SP
authors: Markey
authors: §Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors: Ayse
authors: A
authors: Dosemeci
mesh_terms: D000818:Animals; D013058:Mass Spectrometry / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D013569:Synapses / Q000378:metabolism*
chemical_list: D009419:Nerve Tissue Proteins
keywords: copy numbers; multiple reaction monitoring; postsynaptic density; protein stoichiometry