PolyomX Program, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada T6G 1Z2.
Anal Biochem. 2010 Jan 1;396(1):69-75. doi: 10.1016/j.ab.2009.08.019. Epub 2009 Aug 20.
Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.
蔗糖密度梯度富集膜制剂和通过亲和纯化技术的膜级分富集常用于蛋白质组学分析。然而，通过上述方法表征的已发表蛋白质组学图谱显示除了膜蛋白外还有核蛋白的存在。虽然核蛋白在细胞区室之间穿梭及其在膜界面的瞬时驻留可以解释其中一些观察结果，但在使用粗提和富集膜生成的蛋白质组学图谱中存在核蛋白可能是细胞分馏过程中核碎片非特异性污染的结果。我们假设，由于癌细胞固有的基因组不稳定性而产生的微核可能与蔗糖梯度上的质膜级分共纯化。使用源自 MCF-7 细胞系的乳腺癌细胞系的蔗糖梯度富集质膜，我们提供了实验证据表明微核存在于新鲜的质膜制剂中。这些微核的起源可以追溯到完整细胞中核的出芽。此外，质谱分析证实了蔗糖梯度富集制剂中存在核蛋白以及膜和相关信号蛋白。
