Expanding the human proteome with microproteins and peptideins

PeptideAtlas database construction and searching The human non-HLA PeptideAtlas 2023-06 build contains 295 ProteomeXchange datasets (PXDs) 50 split into 1,172 different experiments that comprise a total of 3.5 billion MS/MS spectra. Sequence database searching was performed using MSFragger 51 v.3.7 using search parameters appropriate for each dataset, depending on alkylation, labelling, fragmentation type, instrument, enrichment strategy and more. All datasets were searched with semi-enzymatic settings (typically semi-tryptic). The search database was 2023-02 THISP level 4 database 52 ( https://peptideatlas.org/thisp/ ), which included the 7,264 Ribo-seq ORFs from ref. 4 as well as other contributed sequences that might be translated. All datasets were searched with generic artifactual variable modifications methionine oxidation, protein N-terminal acetylation, peptide N-terminal pyro-glutamic acid from glutamic acid or glutamine, and asparagine and glutamine deamidation. The alkylation modification was set as a fixed modification (typically carbamidomethylated cysteine). Statistical validation of the results for each experiment was performed using the Trans-Proteomic Pipeline (TPP) 53 , 54 v.7.0 tools PeptideProphet 55 , iProphet 56 and PTMProphet 57 , and the results were mapped to the human proteome using ProteoMapper 58 , taking known variants into account, and to the genome using the ENSEMBL 59 toolkit as previously described 60 . …