Statistical Analysis of Quantitative Peptidomics and Peptide-Level Proteomics Data with Prostar

Prostar is a software tool dedicated to the processing of quantitative data resulting from mass spectrometry-based label-free proteomics. Practically, once biological samples have been analyzed by bottom-up proteomics, the raw mass spectrometer outputs are processed by bioinformatics tools, so as to identify peptides and quantify them, notably by means of precursor ion chromatogram integration. From that point, the classical workflows aggregate these pieces of peptide-level information to infer protein-level identities and amounts. Finally, protein abundances can be statistically analyzed to find out proteins that are significantly differentially abundant between compared conditions. Prostar original workflow has been developed based on this strategy. However, recent works have demonstrated that processing peptide-level information is often more accurate when searching for differentially abundant proteins, as the aggregation step tends to hide some of the data variabilities and biases. As a result, Prostar has been extended by workflows that manage peptide-level data, and this protocol details their use. The first one, deemed "peptidomics," implies that the differential analysis is conducted at peptide level, independently of the peptide-to-protein relationship. The second workflow proposes to aggregate the peptide abundances after their preprocessing (i.e., after filtering, normalization, and imputation), so as to minimize the amount of protein-level preprocessing prior to differential analysis.