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首页> 外文期刊>Molecular & cellular proteomics: MCP >Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.
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Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

机译：通过iTRAQ标记结合在线二维LC / MS / MS对C3H10T1 / 2间充质干细胞进行软骨形成分化的定量蛋白质组学分析。

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The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.
机译：多能间充质干细胞（MSCs）的软骨形成潜力使其成为基于细胞的软骨缺损治疗的有希望的来源。然而，软骨形成以及MSCs自我更新的确切细胞内分子机制仍是未知之数。为了更深入地了解潜在的分子机制，我们将同量异位标签用于相对和绝对定量（iTRAQ）标记，并结合在线二维LC / MS / MS技术来鉴定在软骨形成的体外模型中差异表达的蛋白质：通过微团培养和100 ng / ml骨形态发生蛋白2处理6天，诱导了C3H10T1 / 2细胞（一种小鼠​​胚胎间充质细胞系）的软骨分化。总共鉴定出1756种蛋白质，平均错误发现率<0.21％。定量数据的线性回归分析给出了很强的相关系数：两个重复的二维LC / MS / MS分析的相关系数分别为0.948和0.923，三个独立的iTRAQ实验的相关系数分别为0.881、0.869和0.927（p <0.0001）。在1753个定量的蛋白质中，有100个发生了重大变化（95％置信区间），其中有6个通过蛋白质印迹进一步验证。功能分类显示，这17种上调的蛋白主要包括成熟软骨细胞和参与软骨细胞外基质合成的酶的标志，而下调的83种则主要参与能量代谢，染色质组织，转录，mRNA加工，信号转导和细胞骨架除了许多有据可查的蛋白质外，这些改变的蛋白质中的大多数对于软骨形成都是新颖的。最后，在软骨分化的背景下，验证了BTF3l4和fibulin-5（在本研究中鉴定出的两种新的与软骨形成相关的蛋白）的生物学作用。这些数据将为我们更好地理解调节这些复杂生物过程的潜在机制提供宝贵的线索，并有助于将MSCs应用于基于细胞的软骨再生治疗中。

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