利用化學標定及質譜技術分析經脂多醣刺激後小鼠巨噬細胞 (RAW 264.7) 之比較分泌蛋白質體研究

Abstract

脂多醣(lipopolysaccharide, LPS)是革蘭氏陰性菌細胞壁外膜上一複雜醣脂分子，對於哺乳動物具有毒性，會誘使強烈的發炎反應並產生許多細胞激素，若過度的大量表現，則會導致敗血症和感染性休克。在此篇研究中，我們採用同重元素相對和絕對定量(iTRAQ)的標定策略及質譜技術，分析LPS刺激後小鼠巨噬細胞(RAW 264.7)分泌的蛋白質。為了降低樣品的複雜度並提高鑑定蛋白質的動態範圍以及其序列覆蓋率，在送入奈米級液相層析質譜分析前會先結合強陽離子交換層析法(SCX)或OFFGEL等電聚焦分離技術對iTRAQ標定胜肽進行分離。本實驗共鑑定及定量654種蛋白質，這些蛋白質絕大多數和細胞過程以及調控相關，其中52個蛋白質在LPS刺激後具有顯著表現量的差異。我們從中篩選出經LPS刺激後含量具有顯著上升的蛋白質serglycin進行進一步的研究，利用西方墨點法(Western blot)驗證確認在離體細胞培養液以及活體小鼠血清中證實LPS可以活化serglycin的表現。由於探討LPS刺激RAW 264.7產生的分泌蛋白質的研究不多，因此本研究所鑑定出表現差異蛋白質對於LPS刺激後巨噬細胞產生發炎相關反應提供更多的瞭解。Lipopolysaccharide (LPS) is a complex glycolipid component of outer membrane of gram-negative bacteria. It is highly toxic for mammals, inducing strong inflammatory response and produce many cytokines. In this study, we employed the isobaric tag for relative and absolute quantitation (iTRAQ) labeling strategy and mass spectrometry to analyze the secreted proteins from LPS-stimulated RAW 264.7 cell line. To reduce sample complexity for improving protein dynamic range and sequence coverage, combination of offline strong cation exchange chromatography (SCX) or OFFGEL isoelectric focusing is applied for fractionation of iTRAQ labeled peptides before reversed phase nanoLC mass spectrometry analysis. A total of 654 proteins were identified and quantified; 52 proteins were differentially expressed between LPS-stimulated cells and control cells. Many of them are associated with cellular process and regulation. Serglycin, an up-regulated proteins in LPS-stimulated macrophage cells, was subsequently validated both in vitro (culture medium) and in vivo (mouse serum) by Western blot. This is one of few studies of secretome analysis in LPS-stimulated RAW 264.7 macrophages and these secretory proteins can provide further understanding of LPS-induced inflammation associated responses in macrophages.