Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
J Proteome Res. 2010 Mar 5;9(3):1610-8. doi: 10.1021/pr901138a.
The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior.
人胚胎干细胞（hESC）的大多数分化衍生物缺乏可识别的细胞表面蛋白和相应的抗体，这在很大程度上限制了从混合细胞群中选择特定细胞类型的方法只能采用遗传方法。在这里，我们描述了一种使用基于质谱（MS）的蛋白质组学方法分离 hESC 分化为心肌细胞后的细胞膜蛋白，以鉴定该特定谱系的候选蛋白。定量 MS 区分了心肌细胞特异性的质膜蛋白，这些蛋白在 hESC 和人胎心中分化而来的心肌细胞中高度富集或仅检测到，而在 hESC 衍生的异质分化细胞混合物中则没有检测到。对于几个候选蛋白，通过传统的基于抗体的方法验证了其心肌细胞特异性表达和细胞表面定位。使用针对弹性蛋白微纤维界面蛋白 2（EMILIN2）的抗体，我们证明可以从活细胞群体中对心肌细胞进行分选。除了表明基于 MS 的膜蛋白质组学是一种强大的工具，可用于鉴定候选蛋白以从异质群体中纯化特定细胞谱系外，该方法还生成了一个质膜蛋白质组图谱，提示了控制细胞行为的信号通路。
