以蛋白質體學法分析短鏈脂肪酸代謝異常 ENU 突變鼠
Proteomic analysis of ENU mice with abnormal defect of short chain fatty acid
中文摘要
本研究目的為利用二維電泳法 (Two-dimensional electrophoresis ; 2-DE) 以找出 ENU (ethyl-nitrosourea)突變小鼠表現差異的蛋白質點並進行質譜分析，以瞭解突 變小鼠短鏈脂肪酸代謝異常原因，並且希望能應用此一模式動物於脂肪酸的代謝
研究及探討人類相關臨床疾病。我們利用ENU 注射動物方法藉以誘發大量基因
突變小鼠的產生，經由繁殖配對至第三代後，再以串聯式質譜儀篩得血漿短鏈脂 肪酸 (C4-OH) 代謝異常突變小鼠。實驗小鼠 (C57BL/6J) 分為正常小鼠及血漿 C4-OH 含量較正常小鼠高 4 倍標準差的 ENU 突變小鼠，兩組小鼠分別犧牲後取
其肝臟、肌肉組織，進行粒線體蛋白質萃取後以SDS-PAGE 進行一維電泳，然
而突變小鼠和正常小鼠粒線體蛋白質表現量並無差異。因此，更進一步利用二維 電泳法進行突變小鼠和正常小鼠粒線體蛋白質表現的比較。研究結果顯示突變小 鼠和正常小鼠的肌肉粒線體存有差異的蛋白質點，經由介質輔助雷射脫附游離- 飛行式質譜儀 (Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; MALDI-TOF) 鑑定出差異蛋白質點的身份。在突變小鼠表現量增加 的蛋白質為磷酸化的myosin regulatory light chain 2、tropomysin 1 α chain、
myosin light chain 1，表現量減少的蛋白質為非磷酸化的 myosin regulatory light chain 2、calsequestrin-1 precursor、adenylate kinase isoenzyme 1、ATP synthase D chain。然而這些蛋白質表現的消長是否與突變鼠體內 C4-OH 含量上升有關，仍 有待更進一步的研究加以驗證。關鍵詞：致突變劑 (ethyl-nitrosourea; ENU)、串 聯質譜儀、短鏈脂肪酸、二維電泳法(Two-dimensional electrophoresis ; 2-DE)、介 質輔助雷射脫附游離-飛行式質譜儀(Matrix-assisted laser desorption / ionization time-of-flight mass Spectrometry; MALDI-TOF)
英文摘要
The purpose of this study was to develop a 2-DE coupling with MALDI-TOF analysis to find the differential proteins in ENU mouse which could explore the cause of genetic defect of short chain fatty acid in mice. We hope that the C4-OH animal model can provide us to investigate fatty acid metabolism related to human genetic disease. We generated ENU- mutagenized mouse and applied to MS/MS tandem mass spectrometry to screen third generation progeny of ENU-mutagenized mouse for abnormalities in the pathway of fatty acid metabolism. The short chain fatty acid (C4-OH) level in ENU mice was three to four folds than normal mice. After protein evaluation, there was no differential protein in mitochondria fraction between normal
and ENU mice by SDS-PAGE. Surprisingly, there were eleven differential proteins between normal and ENU mice in muscle in 2-DE approach. These protein spots were applied to MALDI-TOF for futher identification. In results, ENU mouse showed higher protein expressions in phosphorylated myosin regulatory light chain 2, myosin light chain 1 and tropomyosin 1αchain and lower expressions in myosin regulatory light chain 2, adenylate kinase isoenzyme, ATP synthase D chain and calsequestrin-1 precursor in ENU mouse to normal control. These differential proteins expression whether involve to increase C4-OH value in mice needs further study to verify.
Key words：Two-dimensional electrophoresis, ethyl-nitrosourea, C4-OH, MS/MS tandem mass, MALDI-TOF
