Hu Shen, Le Zhang, Newitt Richard, Aebersold Ruedi, Kraly James R, Jones Megan, Dovichi Norman J
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Anal Chem. 2003 Jul 15;75(14):3502-5. doi: 10.1021/ac034154j.
In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the amount of protein present in a single cell is far too small to allow its direct identification by mass spectrometry. In this paper, we report a method by which such proteins can be tentatively identified. We perform standard SDS-PAGE electrophoresis of the proteins contained within a homogenate prepared from an HT29 cell culture. Proteins extracted from bands in the gel are identified by mass spectrometry. The proteins also provide a set of standards that can be used to spike the sample before capillary sieving electrophoresis (CSE) separation; comigration is taken as evidence for the identity of the target protein. In a proof-of-principle experiment, a single band migrating at approximately 47 kDa was isolated from the SDS-PAGE gel generated from the HT29 cell line. Proteins extracted from this band were used to spike a CSE separation of the same extract. This band comigrated with a cell cycle-dependent component identified from single-cell analysis. In-gel digestion and LC/MS/MS were used to identify five proteins, including cytokeratin 18, which is the product of the most highly expressed gene in this cell line.
在本期刊的上一篇论文中，我们报道了使用毛细管筛分电泳来表征单个癌细胞在细胞周期特定阶段所表达的蛋白质。数据分析显示，在99%的置信限下，有一种成分的表达呈现细胞周期依赖性变化。然而，单个细胞中存在的蛋白质数量太少，无法通过质谱直接鉴定。在本文中，我们报道了一种可以初步鉴定此类蛋白质的方法。我们对由HT29细胞培养物制备的匀浆中所含蛋白质进行标准的SDS-PAGE电泳。通过质谱鉴定从凝胶条带中提取的蛋白质。这些蛋白质还提供了一组标准品，可用于在毛细管筛分电泳（CSE）分离之前对样品进行加标；迁移率相同被视为目标蛋白质身份的证据。在一个原理验证实验中，从HT29细胞系产生的SDS-PAGE凝胶中分离出一条迁移率约为47 kDa的单一条带。从该条带中提取的蛋白质用于对同一提取物的CSE分离进行加标。这条带与从单细胞分析中鉴定出的细胞周期依赖性成分迁移率相同。采用胶内消化和液相色谱/串联质谱法鉴定了五种蛋白质，包括细胞角蛋白18，它是该细胞系中表达量最高的基因的产物。
