Schnaars Vanessa, Dörries Marvin, Hutchins Michael, Wöhlbrand Lars, Rabus Ralf
General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University of Oldenburg, Oldenburg, Germany.
Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB), Oldenburg, Germany.
J Mol Microbiol Biotechnol. 2018;28(3):128-136. doi: 10.1159/000494083. Epub 2018 Nov 14.
The efficiency and reproducibility of two-dimensional difference gel electrophoresis (2D DIGE) depends on several crucial steps: (i) adequate number of replicate gels, (ii) accurate image acquisition, and (iii) statistically confident protein abundance analysis. The latter is inherently determined by the image analysis system. Available software solutions apply different strategies for consecutive image alignment and protein spot detection. While DeCyderTM performs spot detection on single gels prior to the alignment of spot maps, SameSpotsTM completes image alignment in advance of spot detection. In this study, the performances of DeCyderTM and SameSpotsTM were compared considering all protein spots detected in 2D DIGE resolved proteomes of three different environmental bacteria with minimal user interference. Proteome map-based analysis by SameSpotsTM allows for fast and reproducible abundance change determination, avoiding time-consuming, manual spot matching. The different raw spot volumes, determined by the two software solutions, did not affect calculated abundance changes. Due to a slight factorial difference, minor abundance changes were very similar, while larger differences in the case of major abundance changes did not impact biological interpretation in the studied cases. Overall, affordable fluorescent dyes in combination with fast CCD camera-based image acquisition and user-friendly image analysis still qualify 2D DIGE as a valuable tool for quantitative proteomics.
二维差异凝胶电泳（2D DIGE）的效率和可重复性取决于几个关键步骤：（i）足够数量的重复凝胶，（ii）准确的图像采集，以及（iii）具有统计学可信度的蛋白质丰度分析。后者本质上由图像分析系统决定。现有的软件解决方案在连续图像对齐和蛋白质斑点检测方面采用了不同的策略。DeCyderTM在斑点图谱对齐之前对单个凝胶进行斑点检测，而SameSpotsTM在斑点检测之前完成图像对齐。在本研究中，在用户干预最小的情况下，比较了DeCyderTM和SameSpotsTM在三种不同环境细菌的2D DIGE解析蛋白质组中检测到的所有蛋白质斑点的性能。SameSpotsTM基于蛋白质组图谱的分析允许快速且可重复地确定丰度变化，避免了耗时的手动斑点匹配。两种软件解决方案确定的不同原始斑点体积并未影响计算出的丰度变化。由于存在微小的因子差异，较小的丰度变化非常相似，而在较大丰度变化的情况下，较大差异并未影响所研究案例中的生物学解释。总体而言，价格合理的荧光染料与基于快速CCD相机的图像采集以及用户友好的图像分析相结合，仍使2D DIGE成为定量蛋白质组学的有价值工具。
