探討層析條件對LC/MS之蛋白質定序偵測操作的影響

Chia Nan University of Pharmacy & Science Institutional Repository > 藥理學院 > 生物科技系(所) > 博碩士論文 > Item 310902800/6168

請使用永久網址來引用或連結此文件: https://ir.cnu.edu.tw/handle/310902800/6168

標題:	探討層析條件對LC/MS之蛋白質定序偵測操作的影響 The Effect of the Chromatographic Parameters Influence the Protein Sequence Analysis of LC/MS
作者:	吳瓊如 Chiung-Ru Wu
貢獻者:	方嘉德嘉南藥理科技大學：生物科技研究所
關鍵字:	液相層析質譜儀修飾劑管柱流速偵測變化考馬斯亮藍 Sypro Ruby Instant Blue Coomassie Brilliant Blue Parameters variables Flow Rate Columns LC/MS
日期:	2005
上傳時間:	2008-10-31 16:15:48 (UTC+8)
摘要:	液相層析質譜儀（LC/MS）目前已經是蛋白質和胜肽分析的重要工具之一，它不僅可以分析蛋白質或胜肽的分子量，也可以提供有關它們的後轉譯修飾如甲基化、磷酸化和醣基化等一系列的相關訊息。可是在分析時，不僅會受到MS參數條件的影響，也會受到層析條件和膠體染色條件等所影響。如染色劑的種類、沖提液的pH值、沖提溶劑的選擇、層析時的流速或是其它如修飾作用等等各項條件。在此實驗中，主要是想找出可以增加蛋白質辦識率的最佳化條件。對SDS-PAGE中蛋白質的偵測可以使用染劑。在染劑的選擇可跟據它是螢光或非螢光，靈敏度，及染色時間的長短來考量。在蛋白質偵測時，由於染劑和蛋白質鍵結的方式與在樣品前處理的過程中是否可完全退染等因素，皆會影響LC/MS的結果。本實驗分別比較了Instant BlueTM Coomassie Blue，考馬斯亮藍（Coomassie Brilliant Blue）和Sypro Ruby對BSA的染色比較。發現Instant Blue與Sypro Ruby的最低偵測結果都是在100ng。由此可知新型的染劑的靈敏度與Sypro Ruby一樣好，但在染色的過程中，卻是操作較為便利，而又容易辨識之。在層析操作時，大部份的蛋白質或胜肽在低pH值的情形下可以被分離出來。修飾劑的使用不僅可以降低沖提液中的pH值，也可以增加分析物在層析管柱中的滯留能力。修飾劑又稱為離子對試劑，因其含有一疏水基可與分析物的非極性部位形成鍵結。而與分析物形成離子對。目前最常被使用的修飾劑有三氟乙酸（trifluoroacetic acid ,TFA），醋酸（acetic acid）或甲酸（formic acid）等。在本實驗中，分別以甲酸、三氟乙酸、五氟丙酸（ pentafluoropropionic acid, PFPA)和七氟丁酸（hyptaflurorobutyric acid, HFBA)來進行比較。結果發現以三氟乙酸為修飾劑時，可以達到10.3%±0.8%的結果；甲酸次之，為9.2%±0.4%；五氟丙酸為7.2%±0.0%，而七氟丁酸為6.05%±1.1%。不同含碳鏈長度的管柱，在分離分析物的能力上也有所不同。碳鏈與支撐物的鍵結形式如monomeric或polymeric也影響著分離能力。碳鏈愈短，所能吸附的胜肽鏈也較短，反之易然。結果發現使用C4的管柱時，可以得到4.9%±1.2%，C8的管柱是7.6%±1.2％，C18的monomeric形式可以得到8%±2.6％，而polymeric則是12.3%±0.4％。Supelco製的C18管柱則是9.8%±2.7%。在進行層析沖提時，流速也是一重要因素。速度若太快則分析物會太快被沖提出來，可能造成辨識上的困難；速度若太慢，已被分離出來的分析物層帶會變寛，不僅在時間上耗費太多，所形成的峰線也不夠尖銳，可能造成峰線拖尾（tailing）的現象，而產生定量偵測的誤差。在實驗中得到了以40μL/min的流速在BSA的辨識率是20.3%±2.3%；20μL/min是21.7%±3.9%；10 μL/min是14.7%±3.6%；5μL/min是24.9%±5.4%。　綜合以上結果，得到最佳化層析條件：使用Instant BlueTM染劑，再採用三氟乙酸為修試劑，沖提液的流速為5μL/min，以及選擇C18 polymeric管柱作為分析管柱。對靈敏度及Real Sample進行測試。在偵測靈敏度方面，以BSA 0.5μg為標的物，分別以2μL/min，0, 5μL/min來進行測試；在20μL/min時，得到4.1%的protein coverage，而在5μL/min時，得到4.9%的protein coverage，兩者各得到二段的胜肽；但是在5μL/min也曾經成弘輕X0.1μg，而得到4.1%的protein coverage，同樣得到二段胜肽。在Real Sample方面，先以β-actin為標的物，進行單盲測試，並且進行比對出正確結果，protein coverage 為36.7%；而後進行雙盲測試，acidic ribosomal phosphoprotein A（樣品），10.5%； prohibitin （B樣品），36.3%；再根據樣品的分子量，等電點（PI）進行比對之後，而確定出這兩種樣品。 LC/MS is used to provide information about protein identifications, molecular weights of proteins or peptides, and post-transcription modification (PTM) of proteins. In the chromatographic analysis, the effects of data will be affected by some conditions, for example, the pH of mobile phase, the solvents, the buffers, the flow rate, the modifiers, the stain reagents, and so on. The goal of this study is to find the best conditions to increase identification of the protein coverage. Proteins separated in the SDS-PAGE will be detected when stain reagents are used. Some parameters which are used to find the best stain reagent for detection include sensitivity, fluorescence or non-fluorescence, and staining time. The type of connection among stain reagents, and proteins, and pre-process sample will influence the effects of the detection of LC/MS. Several kinds of stain regents are compared, including Sypro Ruby, Coomassie Brilliant Blue, silver stain and new stain reagent—Instant BlueTM Coomassie Blue. It is found that the sensitivity is no different when Sypro Ruby or Instant BlueTM Coomassie Blue is used. In the chromatographic analysis, most proteins and peptides are separated at low pH, several organic acids have been used as modifiers, such as trifluoroacetic acid (TFA), acetic, or formic acid. The modifier is an ion-pairing agent. It contains a hydrocarbon chain that will connect with the sample, and it will be non-charge material which will be retained on the reveres-phase column. In this work, the different types of modifiers are compared—formic acid, TFA, PFPA (pentafluroropropionic acid), and HFPA (pentafluoropropionic acid). The results show that formic acid gets 9.2% ±0.4% in coverage; TFA gets 10.3% ±0.8%; PFPA gets 7.2% ±0.0% and HFBA gets 6.05% ±1.1%。 Some types of column including different carbons are compared, too. The stationary phase, for example monomeric (brush) or polymeric (bush), will affect the results of the chromatographic separation. When column with short carbochain is used, it will absorb the short peptides. On the contrary, it will adsorb the long peptides. The results show that column of C4 gets 4.9%±1.2% in coverage; C8 gets 7.6%±1.2％; monomeric type of column gets 8%±2.6％; polymeric type of column gets 12.3%±0.4％ and C18 column made in Supelco gets 9.8%±2.7%。. The flow rate is an important parameter. The flow rate is too fast to separate samples well, and it is hard to identify them. W hen the flow rate is too slow, the separated sample will remix and the peaks will not sharp. This work can help to get optimized conditions for protein analysis. The results show 40uL/min gets 20.3%±2.3% in identified coverage; 20uL/min gets 21.7%±3.9%; 10uL/min gets 14.7%±3.6% and 5uL/min gets 24.9%±5.4%. The best condition which we find is by using following parameters: choice TFA as modifier, C18 polymeric column is used, flow rate is 5μL/min and Instant BlueTM is used. These conditions are applied in the sensitivity and the real sample annlysis. In the sensitivity part, BSA is the target protein and 0.5μg of BSA will be used, and the flow rate is 20 and 5μL/min. We get 4.1% protein coverage with 2 peptides. When flow rate is 20 μL/min, there are 4.9% protein coverage with 2 peptides too. It ever reaches 4.1% when the flow rate is 5μL/min. In the real sample, there are two step we applied. The first is single bland test. The sample isβ-actin. The result is correct. The second is double bland test. There are two samples being chose—Acidic ribosomal phosphoprotein, which we abbreviate as A and prohibitin, which we abbreviate as B. A gets the protein coverage about 10.5%. B gets the protein coverage about 36.3%. According to the molecule weight and pI, the two samples are identified.
關聯:	校內校外均不公開
顯示於類別:	[生物科技系(所)] 博碩士論文

文件中的檔案:

檔案	描述	大小	格式	瀏覽次數
index.html		0Kb	HTML	1656	檢視/開啟

在CNU IR中所有的資料項目都受到原著作權保護.

TAIR相關文章

資料載入中.....