Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132, USA.
J Biotechnol. 2010 Aug 2;148(4):182-93. doi: 10.1016/j.jbiotec.2010.06.005. Epub 2010 Jun 9.
The global turnover rates of cellular proteins and the secretion rate of a recombinant immunoglobulin G (IgG) in a myeloma cell line, NS0, were determined using SILAC proteomic analysis. After complete labeling of cellular proteins with (13)C(6), (15)N(4)-arginine, cells were transferred to unlabeled medium and the decay of the labeled arginine in proteins was monitored during exponential cell growth. After PAGE separation and mass-spectrometric identification of proteins, those detected with high confidence over at least three time points were used for the determination of turnover rates. Among the 224 proteins quantified with a protein half-life, about 15% have a degradation rate constant lower than one-tenth of specific growth rate. For most proteins, the turnover rate is insignificant in its overall dynamics. Only 6.3% of proteins have a half-life shorter than the cell doubling time. For IgG secretion, both heavy and light chain molecules follow the same kinetic behavior with a half-life estimated to be 2h. The label decay curve appears to show a second region with very slow kinetics, raising the possibility of two populations of IgG molecules with different secretion characteristics.
利用 SILAC 蛋白质组学分析，我们测定了骨髓瘤细胞系 NS0 中细胞蛋白质的全球周转率和重组免疫球蛋白 G（IgG）的分泌率。在细胞蛋白质完全用 (13)C(6)、(15)N(4)-精氨酸标记后，将细胞转移到无标记的培养基中，并在细胞指数生长期监测标记精氨酸在蛋白质中的衰减情况。在 PAGE 分离和质谱鉴定蛋白质后，至少在三个时间点被高置信度检测到的蛋白质用于确定周转率。在 224 个用蛋白质半衰期定量的蛋白质中，约 15%的蛋白质降解速率常数低于特定生长速率的十分之一。对于大多数蛋白质来说，周转率在其整体动力学中并不重要。只有 6.3%的蛋白质的半衰期短于细胞倍增时间。对于 IgG 分泌，重链和轻链分子都表现出相同的动力学行为，半衰期估计为 2 小时。标记衰减曲线似乎显示出第二个动力学非常缓慢的区域，这增加了具有不同分泌特征的两种 IgG 分子群体的可能性。
