Updated project metadata. Excessive reactive oxygen species cause oxidative stress in cells. Two molecules of tyrosine can generate dityrosine, which is not only a product of natural posttranslational processes, but also a putative biomarker for protein oxidation. Few studies have investigated dityrosine crosslinking under endogenous or exogenous oxidative conditions in Escherichia coli at the proteome level so far. In this study, in order to investigate qualitative and quantitative dityrosine crosslinking, two mutant strains were constructed as endogenous oxidative stress model, as well as one mutant strain added with 1mM H2O2 as exogenous oxidative stress model. We integrated label-free LC-MS/MS and bioinformatics to research dityrosine crosslinking in Escherichia coli. A large-scale dataset of dityrosine crosslinking was provided. 352 proteins were identified to participate in dityrosine crosslinking by pLink. The subcellular localization included cytoplasmic (61.93%), periplasmic (21.31%), outer membrane (7.95%), inner membrane (5.97%) and extracellular (2.84%). Quantitative analysis of dityrosine crosslinking indicated that the three oxidative stress models could be separated at dityrosine linked peptides level. We expect that our research will guide efforts to comprehend the detailed features of dityrosine crosslinking in future.