Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry:: Mapping the nuclear proteome of human fibroblasts
Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry:: Mapping the nuclear proteome of human fibroblasts
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DOI：
10.1002/elps.200305619
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发表时间：
2003-11-01
期刊：
影响因子：
2.9
通讯作者：
Marko-Varga, G
中科院分区：
文献类型：
--
作者：
Malmström, J;Larsen, K;Marko-Varga, G
介绍了一种结合微凝胶分级分离的微型液相色谱纳米分离方法。我们获得了高灵敏度和高通量鉴定表达的蛋白质的亚细胞分级分离和纳米毛细管液相色谱接口的电喷雾电离（ESI）和基质辅助激光解吸/电离（MALDI）串联质谱。使用在线基质注入，将反相纳米毛细管洗脱液直接施加到MALDI靶板上，形成离散的晶体斑点。当使用原代细胞时，只有有限数量的样品可用。为了最大限度地从有限数量的样品中识别出蛋白质的数量，小型化样品制备方案和纳米流分离是必要的，特别是在处理低丰度蛋白质时。从同一个分离的细胞核样品中，通过二维（2-D）凝胶电泳进行完整蛋白质的互补分离。从核制备物中总共鉴定出594种基因产物，其中261种是独特的。鉴定了几种参与转录事件的蛋白质，例如TATA结合蛋白、EBNA共激活因子和白细胞介素增强子结合蛋白，这表明已经获得了足够的蛋白质组学深度来研究转录控制事件。我们的研究结果表明，通过样品预分馏和缩小的纳米流分离沿着与组合的质谱策略，有可能从人原代细胞中识别出大量的核蛋白。由于这些靶细胞的疾病联系，这些发现特别重要。
Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.