MS-DAP Platform for Downstream Data Analysis of Label-Free Proteomics Uncovers Optimal Workflows in Benchmark Data Sets and Increased Sensitivity in Analysis of Alzheimer’s Biomarker Data

[Image: see text] In the rapidly moving proteomics field, a diverse patchwork of data analysis pipelines and algorithms for data normalization and differential expression analysis is used by the community. We generated a mass spectrometry downstream analysis pipeline (MS-DAP) that integrates both popular and recently developed algorithms for normalization and statistical analyses. Additional algorithms can be easily added in the future as plugins. MS-DAP is open-source and facilitates transparent and reproducible proteome science by generating extensive data visualizations and quality reporting, provided as standardized PDF reports. Second, we performed a systematic evaluation of methods for normalization and statistical analysis on a large variety of data sets, including additional data generated in this study, which revealed key differences. Commonly used approaches for differential testing based on moderated t-statistics were consistently outperformed by more recent statistical models, all integrated in MS-DAP. Third, we introduced a novel normalization algorithm that rescues deficiencies observed in commonly used normalization methods. Finally, we used the MS-DAP platform to reanalyze a recently published large-scale proteomics data set of CSF from AD patients. This revealed increased sensitivity, resulting in additional significant target proteins which improved overlap with results reported in related studies and includes a large set of new potential AD biomarkers in addition to previously reported.