Hu Shen, Le Zhang, Krylov Sergey, Dovichi Norman J
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Anal Chem. 2003 Jul 15;75(14):3495-501. doi: 10.1021/ac034153r.
Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.
细胞周期依赖性蛋白表达的研究在肿瘤学、干细胞研究和发育生物学中具有重要意义。在本文中，我们报告了来自细胞周期中已知阶段的单个细胞的首个蛋白质指纹图谱。为了确定该阶段，我们用活细胞核染料Hoechst 33342处理HT - 29结肠癌细胞。使用显微镜测量一个处理过的细胞的荧光强度；在这种形式的图像细胞术里，荧光强度与细胞的DNA含量成正比，而DNA含量在细胞周期中以可预测的方式变化。为了生成蛋白质指纹图谱，将细胞吸入分离毛细管并裂解。蛋白质用3 -（2 - 呋喃甲酰喹啉 - 2 - 甲醛）进行荧光标记，通过毛细管筛分电泳分离，并通过激光诱导荧光检测。这种电泳形式是SDS - PAGE的毛细管版本。单细胞电泳图谱在30分钟的分离过程中部分解析了大约25个组分，检测器的动态范围超过5000。蛋白质表达存在较大的细胞间差异，整个电泳图谱的相对标准偏差平均为40%。变异的主要来源是细胞在细胞周期中的阶段；平均而言，蛋白质表达中约60%的细胞间差异与细胞周期有关。处于细胞周期G1和G2/M期的细胞蛋白质表达的相对标准偏差分别为27%和21%。如预期即将分裂为两个子细胞的细胞那样，处于G2/M期的细胞产生的信号幅度是处于G1期细胞产生信号的两倍。当将电泳图谱归一化为总蛋白含量时，在99%置信限下只有一种组分的表达依赖于细胞周期。在一篇配套论文中，该蛋白质暂被鉴定为细胞角蛋白18。
