Van Hoof Dennis, Passier Robert, Ward-Van Oostwaard Dorien, Pinkse Martijn W H, Heck Albert J R, Mummery Christine L, Krijgsveld Jeroen
Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Mol Cell Proteomics. 2006 Jul;5(7):1261-73. doi: 10.1074/mcp.M500405-MCP200. Epub 2006 Apr 6.
Embryonic stem cells (ESCs) are of immense interest as they can proliferate indefinitely in vitro and give rise to any adult cell type, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Extensive analyses of numerous human and mouse ESC lines have shown generic similarities and differences at both the transcriptional and functional level. However, comprehensive proteome analyses are missing or are restricted to mouse ESCs. Here we have used an extensive proteomic approach to search for ESC-specific proteins by analyzing the differential protein expression profiles of human and mouse ESCs and their differentiated derivatives. The data sets comprise 1,775 non-redundant proteins identified in human ESCs, 1,532 in differentiated human ESCs, 1,871 in mouse ESCs, and 1,552 in differentiated mouse ESCs with a false positive rate of <0.2%. Comparison of the data sets distinguished 191 proteins exclusively identified in both human and mouse ESCs but not in their differentiated derivatives. Besides well known ESC benchmarks, this subset included many uncharacterized proteins, some of which may be novel ESC-specific markers. To complement the mass spectrometric approach, differential expression of a selection of these proteins was confirmed by Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting. Additionally two other independently isolated and cultured human ESC lines as well as their differentiated derivatives were monitored for differential expression of selected proteins. Some of these proteins were identified exclusively in ESCs of all three human lines and may thus serve as generic ESC markers. Our wide scale proteomic approach enabled us to screen thousands of proteins rapidly and select putative ESC-associated proteins for further analysis. Validation by three independent conventional protein analysis techniques shows that our methodology is robust, provides an excellent tool to characterize ESCs at the protein level, and may disclose novel ESC-specific benchmarks.
胚胎干细胞（ESC）备受关注，因为它们能够在体外无限增殖，并分化为任何一种成体细胞类型，这使其成为再生医学中组织替代的潜在无限来源。对众多人类和小鼠ESC系的广泛分析表明，它们在转录和功能水平上既有普遍的相似之处，也存在差异。然而，全面的蛋白质组分析尚付阙如，或者仅限于小鼠ESC。在此，我们采用了广泛的蛋白质组学方法，通过分析人类和小鼠ESC及其分化衍生物的差异蛋白质表达谱，来寻找ESC特异性蛋白。数据集包括在人类ESC中鉴定出的1775种非冗余蛋白、分化的人类ESC中的1532种、小鼠ESC中的1871种以及分化的小鼠ESC中的1552种，假阳性率<0.2%。数据集比较区分出191种仅在人类和小鼠ESC中鉴定到，而在其分化衍生物中未鉴定到的蛋白质。除了众所周知的ESC标志物外，该子集还包括许多未表征的蛋白质，其中一些可能是新的ESC特异性标志物。为补充质谱方法，并通过蛋白质免疫印迹、免疫荧光共聚焦显微镜和荧光激活细胞分选技术，确认了这些蛋白质中一部分的差异表达。此外，还监测了另外两个独立分离和培养的人类ESC系及其分化衍生物中所选蛋白质的差异表达。其中一些蛋白质仅在所有三个人类系的ESC中鉴定到，因此可能作为通用的ESC标志物。我们的大规模蛋白质组学方法使我们能够快速筛选数千种蛋白质，并选择推定的ESC相关蛋白进行进一步分析。通过三种独立的传统蛋白质分析技术进行验证表明，我们的方法是可靠的，为在蛋白质水平上表征ESC提供了一个优秀的工具，并且可能揭示新的ESC特异性标志物。
