Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, Washington 98195-1700, USA.
J Proteome Res. 2010 May 7;9(5):2412-21. doi: 10.1021/pr901124u.
Lipopolysaccharide (LPS), a glycolipid component of the outer membranes of Gram-negative bacteria, initiates proinflammatory, proapoptotic, and antiapoptotic pathways upon binding to macrophage TLR4. Macrophages that are exposed to LPS become activated and exhibit altered morphology and response to infection. We performed isotope coded affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry to identify proteins that are differently expressed between naive and LPS-activated macrophages. We performed replicate ICAT analyses on RAW 264.7 cultured mouse macrophages as well as C57BL/6 bone marrow derived mouse macrophages. We identified and obtained relative abundances for 1064 proteins, of which we identified 36 as having significantly different expression levels upon activation by LPS. We also compared our results with a two color microarray gene expression assay performed by the Institute for Systems Biology and observed approximately 75% agreement between mRNA transcription and protein expression regarding up- or down-regulation of gene products. We used Western blot analysis to confirm the findings of ICAT and mRNA for one protein, sequestosome 1, the cellular concentration of which was observed to increase upon activation by LPS.
脂多糖（LPS）是革兰氏阴性细菌外膜的糖脂成分，与巨噬细胞 TLR4 结合后启动促炎、促凋亡和抗凋亡途径。暴露于 LPS 的巨噬细胞被激活，表现出形态改变和对感染的反应。我们进行了同位素编码亲和标记（ICAT）、多维液相色谱和质谱分析，以鉴定在未激活和 LPS 激活的巨噬细胞之间表达不同的蛋白质。我们对 RAW 264.7 培养的小鼠巨噬细胞和 C57BL/6 骨髓来源的小鼠巨噬细胞进行了重复的 ICAT 分析。我们鉴定并获得了 1064 种蛋白质的相对丰度，其中我们鉴定出 36 种在 LPS 激活时表达水平有显著差异。我们还将我们的结果与 Institute for Systems Biology 进行的双色微阵列基因表达分析进行了比较，关于基因产物的上调或下调，mRNA 转录和蛋白质表达之间约有 75%的一致性。我们使用 Western blot 分析来验证 ICAT 和 mRNA 的发现，对于一种蛋白质，即自噬相关蛋白 1（sequestosome 1），观察到其在 LPS 激活时细胞浓度增加。
