Capillary sieving electrophoresis/micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells
Capillary sieving electrophoresis/micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells
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DOI：
10.1021/ac0498314
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发表时间：
2004-07-15
影响因子：
7.4
通讯作者：
Dovichi, NJ
中科院分区：
文献类型：
--
作者：
Hu, S;Michels, DA;Dovichi, NJ
我们建立了一种研究哺乳动物单细胞蛋白质表达的二维毛细管电泳方法。第一维毛细管包含SDS-普鲁兰缓冲系统以进行毛细管筛分电泳，其基于分子量分离蛋白质。第二维毛细管含有用于胶束电动毛细管色谱的SDS缓冲液。在6分钟长的初步分离后，将来自第一毛细管的馏分依次转移到第二毛细管，在那里它们通过MECC进行进一步分离。超过100个传输和第二维分离是在一个类似的3.5小时长的时间。我们通过从单个天然MC 3 T3-E1骨祖细胞和用人转录调节因子TWIST转染的MC 3 T3-E1细胞产生蛋白质指纹来证明该技术。我们还提供了MCF-7乳腺癌细胞诱导凋亡治疗前后的单细胞蛋白指纹图。
We have developed a two-dimensional capillary electrophoresis method for the study of protein expression in single mammalian cells. The first-dimension capillary contains an SDS-pullulan buffer system to perform capillary sieving electrophoresis, which separates proteins based on molecular weight. The second-dimension capillary contains an SDS buffer for micellar electrokinetic capillary chromatography. After a 6-min-long preliminary separation, fractions from the first capillary are successively transferred to a second capillary, where they undergo further separation by MECC. Over 100 transfers and second-dimension separations are performed over an similar to3.5-h-long period. We demonstrate this technology by generating protein fingerprints from single native MC3T3-E1 osteoprogenitor cells and MC3T3-E1 cells transfected with the human transcription regulator TWIST. We also present single-cell protein fingerprints from MCF-7 breast cancer cells before and following treatment to induce apoptosis.