Lowenthal Mark S, Markey Sanford P, Dosemeci Ayse
†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.
J Proteome Res. 2015 Jun 5;14(6):2528-38. doi: 10.1021/acs.jproteome.5b00109. Epub 2015 Apr 28.
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.
本文展示了对大鼠大脑皮质突触后致密部（PSD）组分的定量研究，其最终目标是确定PSD复合物中蛋白质的平均拷贝数。使用同位素标记的多肽串联体内部标准物开发了高度特异性和选择性的同位素稀释质谱分析法。PSD蛋白质化学计量学的解释是相对于PSD-95（SAP-90，DLG4）以摩尔比实现的，随后，使用PSD-95的一致文献值估计拷贝数。首次估计了PSD中几种蛋白质的平均拷贝数，包括AIDA-1、BRAGs和致密素的拷贝数。主要发现包括PSD中AIDA-1的高拷贝数证据（144±30），这与整个GKAP蛋白家族的拷贝数（150±27）相当，表明AIDA-1是PSD支架的一个组成部分。对于GluN-‑‑1、GluN2A和GluN2B，NMDA受体亚基的平均拷贝数分别估计为66±18、27±9和45±15，总共产生34±10个NMDA通道口。AMPA通道及其辅助亚基TARPs的估计平均拷贝数分别为68±36和144±38，化学计量比约为1:2，支持了大多数AMPA受体通过TARP亚基锚定到PSD的观点。这种对PSD蛋白质的稳健定量分析改进并扩展了具有指定平均拷贝数的主要PSD组分列表，有助于持续努力揭示PSD复杂的分子结构。
