MSstats Version 4.0: Statistical Analyses of Quantitative Mass Spectrometry-Based Proteomic Experiments with Chromatography-Based Quantification at Scale

[Image: see text] The MSstats R-Bioconductor family of packages is widely used for statistical analyses of quantitative bottom-up mass spectrometry-based proteomic experiments to detect differentially abundant proteins. It is applicable to a variety of experimental designs and data acquisition strategies and is compatible with many data processing tools used to identify and quantify spectral features. In the face of ever-increasing complexities of experiments and data processing strategies, the core package of the family, with the same name MSstats, has undergone a series of substantial updates. Its new version MSstats v4.0 improves the usability, versatility, and accuracy of statistical methodology, and the usage of computational resources. New converters integrate the output of upstream processing tools directly with MSstats, requiring less manual work by the user. The package’s statistical models have been updated to a more robust workflow. Finally, MSstats’ code has been substantially refactored to improve memory use and computation speed. Here we detail these updates, highlighting methodological differences between the new and old versions. An empirical comparison of MSstats v4.0 to its previous implementations, as well as to the packages MSqRob and DEqMS, on controlled mixtures and biological experiments demonstrated a stronger performance and better usability of MSstats v4.0 as compared to existing methods.