Department of Biomedical Engineering, Columbia University, New York, New York, USA.
J Proteome Res. 2011 Jul 1;10(7):3050-9. doi: 10.1021/pr200030v. Epub 2011 Jun 14.
Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering.
我们之前的工作表明，在二维传代过程中用高渗介质处理细胞可使细胞成为软骨组织工程应用的首选。在这里，我们使用无标记的蛋白质组学分析来评估对照和高渗处理环境对多能脂肪干细胞（ASCs）在软骨生成生长因子混合物中培养的表型的影响。在交替的低（前体）和高（产物）碎片化电压（MS(E)）下以数据独立的方式记录光谱。该方法补充了在整个实验过程中在前体离子光谱中进行精确质量和保留时间匹配的精确质量和保留时间匹配的数据分析。结果表明，细胞对渗透压处理有复杂的反应，对照和处理细胞群之间有许多蛋白质表达不同。这些蛋白质中的一些作用在文献中有记载，是所研究的生理状态的特征，特别是醛糖还原酶（渗透胁迫）。该蛋白在本工作中作为阳性对照，提供了独立的佐证验证。其他蛋白质，包括 5'-核苷酸酶和转胶蛋白，以前与细胞分化状态有关。这项研究表明，无标记分析可作为一种有用的工具来描述细胞对软骨生成治疗方案的反应，建议将其用于优化软骨组织工程的细胞启动方案。
