The Escherichia coli PeptideAtlas Build: Characterizing the Observed Escherichia coli Pan-Proteome and Its...

Escherichia coli is a widely used model organism in molecular biology. Despite its pivotal role, a comprehensive proteome resource covering the E. coli pan-proteome and its post-translational modifications (PTMs) has been lacking. Here we present the E. coli PeptideAtlas build, the first comprehensive pan-proteome analysis of E. coli, generated from 40 high-quality public and in-house data sets spanning a broad diversity of strains, sample types, and experimental conditions, and comprising over 73 million MS/MS spectra. All data sets were reprocessed using both a closed search (Trans-Proteomic Pipeline using MSFragger) and an open search (ionbot). The E. coli PeptideAtlas build provides evidence for 4755 proteins, including 1376 previously lacking protein-level support in UniProtKB. The resource offers protein coverage, modification sites, raw spectra with matched peptides, and manually annotated metadata for the E. coli pan-proteome. PTM profiling identified over 10,000 modification sites, including phosphorylation (3806), acetylation (754), methylation (730), glutathionylation (352) and phosphoribosylation (226). Analysis of the glutathionylation sites revealed potential links to metal binding regulation. We also detected proteins likely stemming from phages, underscoring the value of pan-proteomic approaches for studying host-phage interactions. All identifications are publicly accessible and traceable through the PeptideAtlas interface. We expect that the E. coli PeptideAtlas build will provide a useful resource for the community, which supports, for example, targeted MS experiment design, PTM enrichment method development, and strain typing. It allows straightforward lookups of protein and peptide identifications and facilitates comparative proteomic analyses by enabling the assessment of protein presence and variability across different E. coli strains. The build is available at https://peptideatlas.org/builds/ecoli/.