This dataset contains data-independent acquisition (DIA) LC-MS/MS measurements of Escherichia coli samples acquired across a gradient of 13C abundance treatments. The treatment groups correspond to 1%, 2%, 5%, 25%, 50%, and 99% 13C abundance, with three replicate runs collected for each treatment level. The study was designed to characterize proteomic measurements across increasing 13C enrichment conditions and to provide raw mass spectrometry data linked to treatment-specific metadata for downstream analysis and reuse. In total, the dataset includes 18 LC-MS/MS runs acquired on an Astral platform.