Department of Medicine, Division of Respiratory Medicine, Karolinska Institutet, Stockholm, Sweden.
J Proteome Res. 2010 Mar 5;9(3):1522-32. doi: 10.1021/pr9010298.
Historically, the use of two-dimensional electrophoresis (2-DE) in quantitative proteomics has been hampered by significant technical variance. Over the past decade, a range of technological leaps have reduced the overall variance of 2-DE, thus turning the technology into a robust platform for quantitative intact proteomics. However, as the confounding gel-to-gel variation improves, the variance arising from the subsequent image analysis becomes more prominent. Limitations in image alignment and spot detection of previous generations of 2-DE analysis software have demanded considerable user-intervention and manual editing, resulting in introduction of a large degree of subjectivity and software-induced variance. We evaluated the performance of SameSpots, representing a new generation of 2-DE image analysis software, using both DIGE and traditional single-stain 2-DE approaches. Evaluations of the software-induced variance in relation to other sources of variance, as well as the subjectivity through comparison of analyses performed by an expert user and a novice lab-user, were performed. In terms of statistical power, the less-experienced user achieved the better results, but no discernible difference was detected in multivariate comparisons between the users. In conclusion, we found that SameSpots represents improvements both in reproducibility and objectivity in relation to previous generations of 2-DE analysis software.
从历史上看，二维电泳（2-DE）在定量蛋白质组学中的应用受到显著技术差异的阻碍。在过去的十年中，一系列技术飞跃降低了 2-DE 的整体差异，从而使该技术成为定量完整蛋白质组学的强大平台。然而，随着凝胶间差异的改善，随后的图像分析产生的差异变得更加突出。前几代 2-DE 分析软件在图像对齐和斑点检测方面的局限性要求大量的用户干预和手动编辑，导致引入了很大程度的主观性和软件诱导的差异。我们使用 DIGE 和传统的单染 2-DE 方法评估了 SameSpots 的性能，SameSpots 代表了新一代的 2-DE 图像分析软件。我们评估了软件诱导的方差与其他来源的方差之间的关系，以及通过比较专家用户和新手实验室用户执行的分析来评估主观性。就统计功效而言，经验较少的用户获得了更好的结果，但在用户之间的多元比较中没有检测到明显差异。总之，我们发现 SameSpots 在与前几代 2-DE 分析软件相比，在重复性和客观性方面都有所改进。
