Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
Proteomics. 2012 Nov;12(22):3338-42. doi: 10.1002/pmic.201200183. Epub 2012 Oct 19.
The 5-hydroxytryptamine 1A receptor (serotonin 1A receptor; 5-HT(1A) R) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5-HT(1A) R is limited to the presence of two 5-HT(1A) R peptides in rat's whole brain as observed by in-solution digestion followed by LC-MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5-HT(1A) R at the protein level. A rat recombinant 5-HT(1A) R was extracted from the tsA201 cell line, run using several gel-based principles with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin, AspN, proteinase K, and pepsin followed by nano-LC-ESI-MS/MS analysis on a high capacity ion trap and an LTQ Orbitrap Velos. Using two search engines, Mascot and Modiro™, the recombinant 5-HT(1A) R was identified showing 94.55% sequence coverage. A single phosphorylation at S301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5-HT(1A) R, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.
5-羟色胺 1A 受体（血清素 1A 受体；5-HT（1A）R）参与了一系列广泛的大脑功能，并且已经报道了其在焦虑、抑郁和认知中的作用。到目前为止，关于 5-HT（1A）R 的质谱特征的已发表信息仅限于在溶液中消化后通过 LC-MS/MS 观察到大鼠整个大脑中存在两种 5-HT（1A）R 肽。然而，关于蛋白质序列和 PTM 的知识对于产生特异性抗体以及在蛋白质水平上研究 5-HT（1A）R 具有重要意义。从 tsA201 细胞系中提取了大鼠重组 5-HT（1A）R，使用几种基于凝胶的原理进行运行，随后用几种蛋白酶（胰凝乳蛋白酶、胰蛋白酶、AspN、蛋白酶 K 和胃蛋白酶）进行胶内消化，然后进行纳升 LC-ESI-MS/MS 分析在高容量离子阱和 LTQ Orbitrap Velos 上。使用两个搜索引擎 Mascot 和 Modiro™，鉴定了重组 5-HT（1A）R，显示出 94.55%的序列覆盖率。鉴定到一个单一的 S301 磷酸化，并通过磷酸酶处理和一系列氨基酸取代进行了验证。对大脑功能和神经递质的关键参与者 5-HT（1A）R 进行了特征描述，这可能使特异性抗体的产生、未来的设计以及在蛋白质水平上对大鼠的先前研究的解释成为可能。
