近幾年來，以質譜為主的蛋白質體學成長快速並且應用到許多研究領域上。 為了得到在細胞、組織、尿液、血液內的蛋白質定性以及定量的資訊，許多分析策略建立來探索蛋白質體世界，例如同位素親和性標定法，結合了親和性純化方法，來建立分析蛋白質體的一部分，針對只含有半胱胺酸的胜肽進行分析，達到同時定性以及定量蛋白質；結合分段對角層析法，利用胜肽其親、疏水性不同，階段性純化出蛋白質N端，如果使用同位素氘的乙醯化試劑，可以達到同時定性以及定量蛋白質。 為了分析特定蛋白質體的一部分或是針對單一蛋白質進行分析的策略，稱為標的蛋白質體學。

第一個研究主題 (第二章): 二甲基標定的胜肽經由碰撞誘導解離會產生增強的a1離子，而這a1離子可以用來確認蛋白質或胜肽的N端胺基酸。 在此方法中，在蛋白質層次利用同位素氘的甲醛(d2)試劑對未具修飾蛋白質N端以及離胺基酸進行標定，接著進行胰蛋白酶酵素水解。 水解後所生成內部胜肽的自由胺基再與具有醛基的固體支撐物進行還原胺化反應而被抓取。 剩下的部分即是含有二甲基修飾或是在人體內已有修飾的N端胜肽，接著經由液相層析質譜儀進行序列分析。 由於二甲基修飾的胜肽其a1離子的訊號增強且搭配使用氘試劑，可以使得二甲基修飾與人體內已有修飾的N端蛋白質有所區別。 因此，序列以及N端修飾可以藉由二次質譜圖清楚地標示出來。

第二個研究主題 (第三章): 缺氧引發的基因調控會透過組織蛋白的轉譯後修飾變化，而使得染色質重組。 在本研究中，我們利用高準確度質譜來研究組織蛋白的轉譯後修飾，並且定量在缺氧情況下轉譯後修飾的變化。 再者，為了區分轉譯後修飾的變化是否被缺氧誘導因子-1所調控，我們進行在三種生物狀態下(野生型，缺氧誘導因子-1擊低，缺氧誘導因子-1過度表現)海拉細胞的分析。 由於已知組織蛋白H3與缺氧所引發的基因活化或抑制有關，所以我們利用胜肽找出蛋白質的質譜分析策略來詳細地研究由一維膠所純化的組織蛋白H3的甲基化以及乙醯化。

第三個研究主題 (第四章): 穩定二甲基同位素標定對於定量蛋白質體學來說是一個簡單、負擔得起、價格低廉的方法。 二甲基同位素標定的胜肽具有獨特且增強的a1離子，對於全新序列鑑定以及序列驗證很有幫助。 我們建立了一個網站，使用穩定二甲基同位素標定方法應用於蛋白質定量。 透過二甲基同位素標定胜肽的a1離子其理論與實驗值的比較，軟體會驗證序列的正確性，不符合的胜肽會被移除，不列入蛋白質定量。 再者，軟體會利用統計自動由多維液相層析蛋白質鑑定技術所找出的大量蛋白質數據找出沒有變化的蛋白質。 因此，與沒有變化的蛋白質相比，就可以決定出有意義上升或下降變化的蛋白質。

In recent years, MS-based proteomics has been much grown and applied to many research fields. In order to get the information of quality and quantity of proteins contained in the cells, tissues, urine, blood and so on, many analytical strategies have been developed to explore proteomic world such as isotope coded affinity tag (ICAT), which coupled with affinity purification, set up to analyze protein subsets, targeted only cysteine-containing peptides, reached protein identity and quantitation at the same time; COFRADIC (Combined Fractional Diagonal Chromatography), which coupled with stepwise purification, designed to analyze protein subsets, targeted protein N-terminal peptides, achieved protein identity and quantitation if deuterium form acetylation reagent was used. Strategies that were set up to analyze specific protein subsets or single protein were considered to be targeted proteomics.

In the first topic (Chapter 2): Enhanced a1 ion signals were detected upon collision-induced dissociation of dimethyl labeled peptides, can be used to identify the N-termini of proteins or peptides. Dimethyl labeling at the protein level was first performed using formaldehyde-d2 to label all unblocked protein N-termini and lysine residues, followed by trypsin digestion. The free N-terminal amines of internal peptides generated by digestion were captured by solid supports with aldehyde functionalities through reductive amination. The flowthrough fractions, which contained either in vivo or in vitro blocked N-terminal peptides, were subjected to sequence analyses by LC-MS/MS. Owing to the unique feature of a1 signal enhancement associated with dimethylated peptides and the use of the deuterium reagent, the in vitro blocked (or in vivo free) N-termini of proteins could be easily differentiated from the in vivo blocked N-termini.

In the second topic (Chapter 3): Hypoxia induced changes in gene regulation are often a result of changes in chromatin organization which is mediated by variable post-translational modifications of histone proteins. In this study, we report on a high accuracy mass spectrometry-based technique to characterize the global post-translational modifications of histone H3, the most extensively modified histone protein, and to quantify the change of its modification level during hypoxia. Moreover, in order to differentiate whether the hypoxia-induced change is mediated by hypoxia-inducible factor 1 (HIF-1), we conducted the analyses on HeLa cells under three different biological states, wild type, HIF-1 knockdown, and HIF-1 over-expression. We focused on using bottom-up MS approach in detailed characterizations of methylation and acetylation on histone H3 purified from the gel since they are known to correlate with gene activation/repression induced by hypoxia.

In the third topic (Chapter 4): Stable isotope dimethyl labeling is a simple, affordable and popular method for quantitative proteomics. The unique a1 ion enhancement associated with dimethylated peptides is very useful for de-novo sequencing and sequence validation. A validated web server specifically designed for protein quantification using stable isotope dimethyl labeling (PQ-SIDL) is presented. The software validates the sequence identity by matching the experimental and theoretical mass of the dimethylated a1 ion and excludes the un-matched peptides for protein quantification. Moreover, the software automatically deduces a panel of control proteins based on statistics from a combined search result of a large scale data generated from multidimensional protein identification technology (MudPIT). Thus, whether a protein is significantly up- or down- regulated can be determined and annotated in the result output based on statistical ratio comparison with the control.

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