Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore.
Proteome Sci. 2007 Sep 26;5:18. doi: 10.1186/1477-5956-5-18.
Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).
Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.
The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.
在恒定电场的影响下，穿过交联聚丙烯酰胺凝胶（PAG）的蛋白质会经历扩散和非特异性滞留在凝胶基质中等负面因素。这些负面因素会降低在定义的凝胶体积内的蛋白质浓度，随着迁移距离的增加，从而降低蛋白质分离效率。通过实施脉冲场反转凝胶电泳（FIGE）来提高蛋白质分离效率。
在不同 PAG 百分比下，比较了 FIGE 和恒场电泳（CFE）对模型蛋白质物种和大蛋白质复合物的分离效果。尽管运行时间较长，但具有适当正向和反向脉冲时间比的 FIGE 中的蛋白质条带强度优于 CFE。这些结果显示出每定义凝胶体积的带强度增加。在高达 14%的 PAG 百分比下观察到蛋白质相对迁移率的双峰相移。然而，在更高的 PAG 百分比下，FIGE 对蛋白质分离的影响是随机的。与 CFE 相比，在二维聚丙烯酰胺凝胶电泳（2D PAGE）的二维分离中，大鼠肝裂解物经 FIGE 处理后，可分辨斑点的数量增加了 20%。从 FIGE 和 CFE 中都选择了 9 个常见斑点进行质谱（MS）肽测序，结果表明 FIGE 中所有 9 个蛋白质斑点的最终离子得分更高。与 CFE 相比，使用 FIGE 可以明显看出 800 kDa 到 2000 kDa 以上的天然蛋白质复合物。
本研究表明，在适当的条件下，FIGE 通过增加局部蛋白质浓度，提高了 PAGE 过程中的蛋白质分离效率。FIGE 可以在任何实验室环境中，通过最小的额外仪器实现。尽管运行时间较长，但 FIGE 仍然是一种强大的蛋白质分离工具。
