Trinidad Jonathan C, Specht Christian G, Thalhammer Agnes, Schoepfer Ralf, Burlingame Alma L
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.
Mol Cell Proteomics. 2006 May;5(5):914-22. doi: 10.1074/mcp.T500041-MCP200. Epub 2006 Feb 1.
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 approximately 80% of identified phosphorylation sites are novel.
在哺乳动物的中枢神经系统中，被称为突触后致密物（PSD）的结构是一种蛋白质密集复合体，其功能是检测并响应从突触前轴突终末释放的神经递质。该分子机制中蛋白质磷酸化的调节对于其组成成分的活性至关重要，这些成分包括神经递质受体、激酶/磷酸酶、支架分子以及调节细胞骨架结构的蛋白质。为了表征PSD样品中蛋白质的磷酸化状态，我们将强阳离子交换（SCX）色谱法与固定化金属离子亲和色谱（IMAC）相结合。最初，胰蛋白酶肽段通过阳离子交换进行分离，并通过与串联质谱联用的反相色谱进行分析，这使得在大多数SCX馏分中鉴定出了磷酸化肽段。由于这些单独的馏分中的每一个都过于复杂，无法在单次液相色谱 - 串联质谱（LC-MS/MS）分析中完全表征，我们通过对每个SCX馏分进行IMAC来富集磷酸化肽段，相对于单独使用任何一种方法（SCX或IMAC），鉴定出的磷酸化肽段增加了至少3倍。这使我们能够在突触后致密物制剂中发现的所有蛋白质的23%（1264个中的287个）上鉴定出至少一个磷酸化位点。总共，我们鉴定出998个独特的磷酸化肽段，对应于723个独特的磷酸化位点。在所有磷酸化肽段的62%（998个中的621个）上确定了至少一个精确的磷酸化位点，并且大约80%的鉴定出的磷酸化位点是新发现的。
