Manuscript abstract Noncanonical epitopes presented by Human Leucocyte Antigen class I (HLA-I) complexes to CD8+ T cells attracted the spotlight in the research of novel immunotherapies against cancer, infection and autoimmunity. Proteasomes, which are the main producers of HLA-I-bound antigenic peptides, can catalyze both peptide hydrolysis and peptide splicing. The prediction of proteasome-generated spliced peptides is an objective that still requires a reliable (and large) database of non-spliced and spliced peptides produced by these proteases. Here, we present an extended database of proteasome-generated spliced and non-spliced peptides, which was obtained by analyzing in vitro digestions of 80 unique synthetic polypeptide substrates, measured by different mass spectrometers. Peptides were identified through invitroSPI method, which was validated through in silico and in vitro strategies. The peptide product database contains 16,631 unique peptide products (5,493 non-spliced, 6,453 cis-spliced and 4,685 trans-spliced peptide products), and a substrate sequence variety that is a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing. Potential artefacts and skewed results due to different identification and analysis strategies are discussed. File description PBdataset-search-results.zip Mascot search results for the PB dataset, used for invitroSPI analysis of PB dataset. Contains a full list of all PSMs suggested by Mascot. ProteasomeDB.csv Large database of proteasome-generated spliced and non-spliced peptides, generated by invitroSPI analysis of 80 synthetic polypeptide substrates. databases.zip Custom databases used for search of in vitro digestion MS measurements. They contain all theoretically possible spliced and non-spliced peptides that could be derived from a given polypeptide. analysis-scripts-figshare.zip Scripts used for the analysis presented in the corresponding manuscript. invitroPB_scripts.zip Scripts for the invitroPB method. Online-Table 1 Technical details of all 80 synthetic substrates and their measurement. Online-Table 2 PSMs identified in the gp100 Fusion dataset Online-Table 3 PSMs identified in the PB dataset Online-Figure 1 Comparison of measured MS2 spectra of a representative pool of identified non-spliced and spliced peptide products and synthetic peptides derived from the gp100 dataset Online-Figure 2 Comparison of measured and predicted MS2 spectra of non-spliced and spliced peptides identified by invitroSPI method in the PB dataset Online-Figure 3 Comparison of measured and predicted MS2 spectra of non-spliced and spliced peptides identified by invitroPB method in the PB dataset