An MSstats workflow for detecting differentially abundant proteins in large-scale data-independent acquisition mass spectrometry experiments with FragPipe processing

Technological advances in mass spectrometry and proteomics have made it possible to perform larger-scale and more-complex experiments. The volume and complexity of the resulting data create major challenges for downstream analysis. In particular, next-generation data-independent acquisition (DIA) experiments enable wider proteome coverage than more traditional targeted approaches but require computational workflows that can manage much larger datasets and identify peptide sequences from complex and overlapping spectral features. Data-processing tools such as FragPipe, DIA-NN and Spectronaut have undergone substantial improvements to process spectral features in a reasonable time. Statistical analysis tools are needed to draw meaningful comparisons between experimental samples, but these tools were also originally designed with smaller datasets in mind. This protocol describes an updated version of MSstats that has been adapted to be compatible with large-scale DIA experiments. A very large DIA experiment, processed with FragPipe, is used as an example to demonstrate different MSstats workflows. The choice of workflow depends on the user's computational resources. For datasets that are too large to fit into a standard computer's memory, we demonstrate the use of MSstatsBig, a companion R package to MSstats. The protocol also highlights key decisions that have a major effect on both the results and the processing time of the analysis. The MSstats processing can be expected to take 1-3 h depending on the usage of MSstatsBig. The protocol can be run in the point-and-click graphical user interface MSstatsShiny or implemented with minimal coding expertise in R. Technological advances in bottom-up mass spectrometry-based proteomics have resulted in a substantial increase in the volume and complexity of the resulting data, and for comparative studies, large numbers of samples are required to get statistically meaningful results.MSstats can be used to perform statistical analysis of the data after the peptides and proteins have been identified and quantified. MSstatsBig is a variant specifically designed to manage very large datasets. MSstats is used for the statistical analysis of proteomics data from DIA mass spectrometry experiments. This protocol describes workflows using (i) the MSstatsShiny graphical user interface and (ii) the R packages MSstats or MSstatsBig (for larger datasets).