| dc.description.abstract | Human embryonic stem cells (hESCs) are pluripotent cells that are uniquely capable of differentiation and self-renewal and have a great potential in regenerative medicine and drug discovery. Membrane proteins have been investigated to play important roles in regulating ESC functions and expected to be extensively used for stem cell classification and purification and monitored of the differentiation stages. For example, the NOTCH receptor, a cell surface protein of hESCs, will translocate from membrane to nucleus when it is activated and further differentiated toward the neural fates. As a result, we hypothesize that a comprehensive quantitative analysis of the subcellular proteome, including membrane, cytosolic and nuclear fractions between hESCs and differentiated cells, will help to identify not only new cell surface markers but also molecules involving in regulation of ES functions. However, due to the difficulties in maintenance and culturing of hESCs, very limited cell number can be obtained for proteome analysis.
To overcome this challenge, we aim to develop a sensitive quantitation strategy integrating a micro-fractionation method for quantitative proteomic analysis of the low sample amount of stem cells. StageTips (Stop and Go Extraction Tips) have been reported to provide separation of a few micrograms of sample with high efficiency, recovery and reproducibility. After StageTip fractionation, the number of protein increased from 445 to 765 and 902 by using SCX-StageTip and SAX-StageTip fractionation respectively. In addition, we compared the SAX-StageTip and SCX-StageTip using membrane proteins purified from HeLa cells. A total of 623 proteins (59.7 %) are commonly identified. Regarding the identification number, SAX-StageTip fractionation can obtain 17.9% higher number than SCX-StageTip fractionation. We applied SAX-StageTip fractionation with iTRAQ quantitation strategy on hESC subcellular proteomics. 6236, 4234 and 2061 proteins were identified, of which 3125, 1408 and 865 proteins were successfully quantified in nucleus, cytosol and membrane fraction, respectively. Several differentially expressed proteins had been reported as specific hESC markers and EB markers. In addition, there were 192 differentially expressed plasma membrane proteins which may be considered as potential hESC markers or EB markers. Several differentially expressed proteins would translocate from different cellular localization such as -catenin which have been reported to regulate hESC pluripotency with translocation from cytoplasm into nucleus. Our strategy can not only help to discover the specific marker candidates for hESC but may delineate the mechanism of self-renewal and differentiation. | en_US |