This dataset contains label-free DIA LC-MS/MS measurements of controlled bacterial spike-in samples designed to evaluate quantitative performance across a wide dynamic range. The samples consist of defined mixtures of 13C-labeled E. coli peptides spiked into unlabeled backgrounds at different ratios (2%, 5%, and 50%) and dilution levels (1/10 and 1/100), along with blank controls. Each condition includes three biological replicates, resulting in a total of 21 LC-MS/MS runs. The dataset was generated to benchmark the sensitivity, quantitative accuracy, and dynamic range of Orbitrap Astral mass spectrometry for metaproteomic and stable isotope probing applications. The experimental design enables systematic evaluation of isotope labeling detection, quantitative linearity, and performance under varying abundance conditions.