Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97553
標題: 1.快速、簡便及高通量極致液相層析串聯質譜儀方法搭配正負離子化切換於丹參有效成分之分析 2.快速微波輔助策略降低胰蛋白酶水解胜肽圖譜分析過程中人為之去醯胺化
1.A Rapid, Simple, and High Throughput UPLC-MS/MS Method for Simultaneous Determination of Bioactive Constituents in Salvia Miltiorrhiza with Positive/Negative Ionization Switching 2. Rapid Microwave-Assisted Procedures to Reduce Artificial Deamidation during Trypsin Peptide Mapping
作者: 林庭嫻
Ting-Sian Lin
關鍵字: 丹參;脫胺;液相層析串聯質譜儀;Salvia Miltiorrhiza;Deamidation;UPLC-MS/MS
摘要: 
1.丹參(Salvia miltiorrhiza)為一種常見在亞洲地區做為中醫處方,根據文獻指出丹參中所含對人體有功效化合物可分兩大類,其一是含有水溶性的酚酸類化合物,其二為脂溶性丹參酮類化合物,在亞洲國家已被廣泛應用於臨床上大約兩千年。而目前萃取與分析丹參中酚酸類及丹參酮類的方法耗時且缺乏效率,因此本研究目的為開發新的檢測平台且具有快速分析及簡易樣品萃取優勢,針對丹參植物的萃取化合物利用超高壓液相層析儀器搭配三段式四極棒質譜儀 (ultrahigh-performance liquid chromatography tandem mass spectrometry, UPLC-MS/MS) 做為分析儀器,樣品經由分析管柱 Kinetex F5 column (2.1 x 100 mm, 1.7 μm, pentafluorophenyl) 使用 78% acetonitrile 含有 0.1% formic acid 為動相溶液,採用流速為 0.45 mL/min 等梯度模式,該分析在質譜搭配選擇反應偵測模式 (multiple reaction monitoring,MRM) 與設定正負電掃描切換模式 (polarity switching),可以 2 分鐘內快速分離 8 種有效化合物,此定量方法的線性濃度範圍為 3-3200 ng/mL 且具有良好的線性曲線 (r2 > 0.99),最低偵測極限 (limit of detection, LOD) 與最低定量極限 (limit of quantification, LOQ) 分別 0.023-0.75 ng/mL 以及 0.375-1.5 ng/mL。本分析方法確效在 intra-day 和 inter-day 精密度 (precisions) 相對標準偏差 (relative standard deviation, RSD) 在 15% 內,準確度 (accuracy) 範圍在 93%~118%,丹參的樣品萃取效率 (extraction efficiency) 大於 87%,基質效應 (matrix effect) 為 84~94%。採用此 UPLC-MS/MS 條件的分析時間只需要兩分鐘,便可成功分離丹參中常見酚酸類和丹參酮類,且與文獻相比之下此高通量分析方法條件為最時間最短,同時有效減少有機溶劑的消耗量,並期許未來以此方法來分析丹參中有效化合物含量並提供育種者進行丹參育種之依據。
2.天冬醯胺 (asparagine, Asn) 的脫胺作用會從 asparagine (Asn) 轉變成天門冬胺酸 (aspartate, Asp)是生物體中常見的蛋白質轉譯後修飾,尤其在蛋白質藥物常會出現此作用,導致蛋白質結構改變影響其活性及藥理作用,因此有效地進行準確的定量備受期望。液相層析儀-串聯式質譜儀 (LC-MS/MS) 為強大的分析工具。現今分析方法為將蛋白質藥物進行酵素水解 (digestion) 的過程中,易造成 Asn 脫胺因此無法準確判斷是來自製藥過程或是品管處理過程。本次的研究目的為利用微波輔助酵素水解來建立一套可大幅降低 asparagine 脫胺反應於酵素水解過程中。首先比較兩種實驗室常用的緩衝溶液 Tris buffer 與 ABC buffer,以 標準品PENNY peptide (GFYPSDIAVEWESNGQPENNYK) 作為平台開發,結果顯示 Tris buffer 其脫胺情況減少 52.23%。進一步比較微波輔助水解法與傳統水解方法,其高功率微波和低功率微波脫胺分別減少 32.1% 和 37.1%,微波輔助皆顯著改善脫胺情況。後續將真實樣品 (Recombinant monoclonal antibody, mAb) 利用微波輔助水解法在高解析度串聯式質譜儀進行序列覆蓋率分析其結果為 36.3~38.7%,並從中找到一段 Marker peptide (VNSAAFPAPIEK→ VDSAAFPAPIEK) 與傳統方法比較之下,高功率微波脫胺減少 82.22%、低功率則下降了54.76%。因微波水解易發生錯失切位胜肽,故藉由線性離子阱質譜儀之裂解方法-電子轉移裂解 (Electron transfer dissociation, ETD),結合 CID 與 ETD 之序列覆蓋率為 41~42%。綜合上述結果指出脫胺反應是與pH值改變以及水解反應時間有很大的關係,本篇利用微波水解可縮短水解反應時間並能有效降低 asparagine 脫胺作用,未來希望將此酵素水解平台可應用於蛋白藥物品管上亦可應用於其它蛋白質之 Asn 脫胺作用之相關研究。

1.Salvia miltiorrhiza has account of the medical value, owing to it contains high level of salvianolic acid and tanshinones, and has been widely used in clinic for more than 2,000 years in Asia[1]. However, the conventional approach for the extraction and analysis of phenolic acids and tanshinones from Salvia miltiorrhiza was time-consuming and inefficient. The goal of this study was to develop a new fast and simple ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) analytical method for the analysis of phenolic acids and tanshinones in Salvia miltiorrhiza. The samples were separated on a Kinetex F5 column (2.1 x 100 mm, 1.7 um, pentafluorophenyl). The analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) with polarity switching mode. The linearity of the calibration curve of eight compounds were in the range from 3-3200 ng/mL (r2 > 0.99). Method validation was performed in terms of linearity, the relative standard deviation (RSD) of the intra-day and inter-day, precisions were within 15 % and the accuracy ranged from 93% to 118%. The extraction efficiency was more than 87 %. The limit of detection (LOD) and limit of quantification (LOQ) of the eight analytes varied between 0.023 to 0.75 ng/mL and 0.375 to 1.5 ng/mL, respectively. This approach has the shortest analysis time for the separation of phenolic acids and tanshinones in Salvia miltiorrhiza using UPLC core-shell column with F5 stationary phase. Meanwhile, reducing consumption of organic solvents and total run time (2 min) with high throughput.
2.Deamidation is a common protein post-translational modification, it can convert asparagine (Asn) into aspartate (Asp), and it's also a common degradation mechanism of protein pharmaceuticals. The changes in protein structure occurs when deamination modifies in the active part of protein residue. Since deamidation has negative impacts on protein pharmaceuticals, accurate quantitation of Asn deamidation in protein pharmaceuticals is highly demanded. Owing to the fast development of mass spectrometry, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an powerful analytical platform in decades. In recent years, microwave-assisted trypsin digestion can reduce the time of sample preparation. Thus, in this study, the purpose is to establish a PENNY peptide of trypsin digestion by using microwave-assisted method. In this study, two commonly used ABC and Tris buffers that were optimizaed for trypsin activity. The results demonstrated that lower levels of Asn deamidation were observed in Tris buffer (reduce 63.47%). Next, deamidation levels during reduction and alkylation were evaluated in high and low power microwave, which reduced 32.1% and 37.1%, respectively. Then, monoclonal antibody (mAb) was analyzed by using high-resolution MS, and the sequences coverage of mAb was 38.7~46%. Asn deamidation level of the target peptide (VNSAAFPAPIEK→ VDSAAFPAPIEK) from digestion of the mAb in 20 mM Tris was measured, which reduced 82.22% and 54.76 % in the high and low power microwave, respectively. Owing to microwave-assisted digestion is easy to produce miss cleavage of peptides. It was analyzed by using low-resolution MS coupled with collision-induced dissociation (CID) and electron transfer dissociation (ETD). The result showed that sequences coverage of mAb was 41~44%. Above all, the proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures. In future, this platform can be applied on the quality control of protein pharmaceuticals, and help the researches about Asn deamination.
URI: http://hdl.handle.net/11455/97553
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-14起公開。
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