張煥正林震煌謝芝茜Chih-Chien Hsieh2019-09-042017-6-182019-09-042012http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0699420663%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/100963分析的葡萄糖胺聚醣 (glycosaminoglycans，GAGs)是做為診斷黏多醣症 (mucopolysaccharidosis disease，MPS)及判斷治療效果的重要依據。本篇利用微波加熱輔助進行GAGs降解，搭配聚精胺酸奈米鑽石探針進行純化再結合基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)達到可高通量、定性、定量分析小體積生物樣品中的硫酸化GAGs。利用微波加熱進行GAGs甲基化(Methanolysis)降解,例如硫酸軟骨素多醣 (chondroitin sulfate, CS)甲基化降解，能有效的將反應時間由75分鐘縮短到7分鐘。而利用本實驗室之前所發展出覆蓋著聚精胺酸的奈米鑽石探針，可藉由聚精氨酸與硫酸化多醣之間的高專一性結合特性，使其能夠在高稀釋溶液、尿液中專一性的萃取出CS，達到快速純化進入MALDI MS分析。我們藉由配置同位素甲基化試劑來降解CS做為內標準品，成功的克服了MALDI因雷射功率及非勻相結晶所導致訊號的低再現性，定量曲線的線性範圍從50-2.5 ng，並且具有好的線性(R2 = 0.999)。本篇所使用的基質NEDC其基質訊號干擾小，適合用來分析降解後CS，且高耐鹽，因此分析在複雜且含有高鹽類環境中(ex.尿液、腦脊髓液)之CS，可不需經由去鹽的步驟就可直接進入MALDI進行分析。綜合以上，我們成功的發展出快速(30 分鐘內)且靈敏(LOD =75 pg)用來偵測複雜環境中GAGs的方法，可有效的運用在診斷MPS及監控治療效果上。Degraded fragments of sulfated glycosaminoglycans (GAGs) are key reporters for profiling the burden of mucopolysaccharidosis (MPS) disease at baseline and during therapy. Here, we present a high-throughput assay, which combines microwave-assisted GAGs degradation, polyarginine affinity purification, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), for quantitative analysis of sulfated oligosaccharides in biological samples. First, sulfated oligosaccharides were selectively isolated from highly diluted solutions, spiked urine and spiked artificial Cerebrospinal Fluid (aCSF) using polyarginine-coated nanodiamonds (PA-coated NDs) as a solid phase extraction support. Second, they were degraded through microwave-assisted methanolysis, which significantly reduced the reaction time from hours to minutes. To achieve quantitative analysis, the GAG such as chondroitin sulfate (CS) was mixed with an internal standard derived from its deuterated methanolysate and then analyzed by MALDI MS using N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) as the matrix. The calibration curve of this MS-based analysis showed a linear range from 50 ng to 2.5 ng with a correlation coefficient of 0.999. The new assay is rapid (less than 30 min) and sensitive (with a detection limit of 75 pg), and is potentially useful for clinical diagnosis of MPS.葡萄糖胺聚醣黏多醣症基質輔助雷射脫附游離飛行時間質譜儀奈米鑽石GAGMPSMALDI-TOF MSnanodiamond