Region-based statistical analysis of 2D PAGE images

A new comprehensive procedure for statistical analysis of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) images is proposed, including protein region quantification, normalization and statistical analysis. Protein regions are defined by the master watershed map that is obtained from the mean gel. By working with these protein regions, the approach bypasses the current bottleneck in the analysis of 2D PAGE images: it does not require spot matching. Background correction is implemented in each protein region by local segmentation. Two-dimensional locally weighted smoothing (LOESS) is proposed to remove any systematic bias after quantification of protein regions. Proteins are separated into mutually independent sets based on detected correlations, and a multivariate analysis is used on each set to detect the group effect. A strategy for multiple hypothesis testing based on this multivariate approach combined with the usual Benjamini–Hochberg FDR procedure is formulated and applied to the differential analysis of 2D PAGE images. Each step in the analytical protocol is demonstrated by using an actual dataset. The effectiveness of the proposed methodology is shown using simulated gels in comparison with the commercial software packages PDQuest and Dymension. We also introduce a new procedure for simulating gel images.