The Size-Exclusion Chromatography Algorithmic Toolkit (SECAT) implements a novel, network-centric computational framework for differential PPI network analysis. In this study, we extend co-fractionation / mass spectrometric data analysis to study PPI network dynamics, thus enabling qualitative and quantitative proteome organization assessment across samples and states. We and others have previously shown that chromatographic fractionation of native protein complexes in combination with bottom-up mass spectrometric analysis of consecutive fractions supports the multiplexed characterization and detection of state-specific changes of protein complexes. They are essential for macromolecular complex formation, which in turn constitutes the basis for protein interaction networks that determine the functional state of a cell. Protein-protein interactions (PPIs) play critical functional and regulatory roles in cellular processes. (g) Consistency of PPI detection between replicates of the same conditions. Solid lines represent the true, whereas dashed lines represent the false PPIs. (f) The sensitivity of PPI detection in dependency of SECAT q-value for different levels of reference PPI network accuracy is depicted. For the dilution 1:0, no a priori false PPIs are tested, thus the true FDR equals to 0. (e) The SECAT estimated q-value is accurate as evaluated against the ground truth for all levels of reference PPI network accuracy. (d) The SECAT score histograms depict the dilution of the reference PPI network (true: CORUM) with negative PPIs (false: CORUM-inverted) for the benchmark ( STAR Methods). Numbers in brackets indicate ROC area under the curve values for each partial or combined score. specificity of the different SECAT partial scores, the kickstart score and the integrated SECAT score. (c) The receiver-operating characteristic (ROC) illustrates the sensitivity vs. Colors indicate different peak-picking options line types indicate number of detections with the three replicates per condition. The PPI were filtered to a global context q-value<0.05. (a) The effect of different peak-picking methods for signal processing on peak-width and PPI quantification within replicates of the same experimental condition is depicted in violin plots (lines representing 25, 50 and 75% quantiles respectively, STAR Methods). Signal processing and PPI detection performance evaluated using the Jurkat-Fas ( Scott et al., 2017) dataset.
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