Chapter I.
局部轉譯在軸突中，對於維持以及各種功能上扮演著重要的角色，而軸突中局部轉譯的功能不良亦牽涉到許多神經系統性的疾病。異質性核醣核酸蛋白質(hnRNPs) 是核糖核酸 (RNA) 的結合蛋白質，在RNA代謝中具有多種功能。在此篇研究中，我們藉由整合後的大鼠蛋白質體數據庫分析比對已發表的軸突質譜鑑定數據庫，指出在培養的大鼠大腦皮質神經元細胞中的軸突具有20種hnRNPs。在鑑定到的hnRNPs中，hnRNPs Q和R之間的序列是具有高度相似性。同時，反轉錄聚合酶鏈式反應結果顯示軸突裡的hnRNPs Q和R的mRNAs含量並不高。我們更進一步發現在BDNF刺激下，會提升神經細胞及軸突中hnRNPs Q和R蛋白質的表現量。此外，BDNF的刺激亦會增加軸突中poly(A) RNA的表現量以及含有hnRNPs Q和R的poly(A) RNA顆粒的比例。然而，在切斷神經細胞本體與軸突之間的連結後，BDNF仍可以刺激被切斷後的軸突之局部轉譯，如同它能刺激完好的軸突一樣，但是BDNF卻無法提高軸突中hnRNPs Q和R蛋白質的表現量、poly(A) RNA的含量以及poly(A) RNA與hnRNPs Q和R的colocalization。此結果也與BDNF可增加RNA顆粒在軸突中傳遞運送的結果一致，更進一步顯示BDNF會藉由增加軸突上RNA的運送來調控hnRNPs Q和R的表現量。
Chapter II:
在已存在的高含量蛋白質中鑑定新生成蛋白質的合成是一項具有挑戰性的研究。為了克服這個困難，我們嘗試利用多重反應監測質譜分析（MRM-MS）並結合脈衝標記定量蛋白質組學分析（pSILAC)，在短時間內使用胺基酸進行穩定同位素的標定，做為研究新生成蛋白質合成的方法。相較於傳統的全掃描式質譜分析，多重反應監測質譜分析的特色是只在所選擇的候選胜肽之特定的母離子中的質荷比範圍內做掃描，減少了其他離子信號的干擾，以提高檢測的靈敏度和準確度。本篇研究中，我們選擇了72種候選蛋白質，並預先使用神經母細胞瘤N2a (Neuro-2a) 細胞，在經過2小時的新生成蛋白質之標定後，收集其蛋白質來測試此方法的可能性。 隨後，我們更進一步將此方法應用於神經細胞與軸突的新生成蛋白質之鑑定。從初步結果來看，在神經細胞和軸突中可以分別鑑定到16種和1種蛋白質。

Chapter I:
Local translation plays important roles in the maintenance and various functions of axons, and dysfunctions of local translation in axons are implicated in various neurological diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA binding proteins with multiple functions in RNA metabolism. Here, we identified 20 hnRNPs in the axons of cultured rat cortical neurons by interrogating published axon mass spectrometric databases with rat protein databases. Among those identified in axons are highly related hnRNPs Q and R. RT-PCR analysis indicated that axons also contained low levels of hnRNPs Q and R mRNAs. We further found that BDNF treatments raised the levels of hnRNPs Q and R proteins in whole neurons and axons. BDNF also increased the level of poly(A) RNA as well as the proportion of poly(A) RNA granules containing hnRNPs Q and R in the axon. However, following severing the connection between the cell bodies and axons, BDNF did not affect the levels of hnRNPs Q and R, the content of poly(A) RNA, or the colocalization of poly(A) RNA and hnRNPs Q and R in the axon any more, although BDNF still stimulated the local translation in severed axons as it did in intact axons. The results are consistent with that BDNF enhances the axonal transport of RNA granules. The results further suggest that hnRNPs Q and R play a role in the mechanism underlying the enhancement of axonal RNA transport by BDNF.
Chapter II:
Measuring the synthesis of new proteins in the high-abundant of pre-existing proteins is challenging. To overcome this obstacle, we tried to perform a combination method of multiple reaction monitoring mass spectrometry (MRM-MS) assay and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) to study protein synthesis. In order to increase the sensitivity and accuracy of detection, selected peptide’s transitions of MRM which scan across narrow m/z range instead of conventional mass spectrometry was utilized in this study. A total of 72 candidate proteins were selected. Subsequently, labeling of synthesized proteins obtained from N2a cells within 2hrs had first been used to test the method. I further applied this method to detect the de novo proteins in whole neurons and axons. From preliminary results, the local syntheses of 16 and 1 proteins in whole neurons and axons, respectively, were evaluated.

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