This dataset contains processed files generated in the analyses described in "Discovery of protein modifications using high resolution differential mass spectrometry proteomics" by Cifani, Zhi, Luo et al., describing SAMPEI, a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Using synthetic standards and controlled chemical labeling experiments, we demonstrate high specificity and sensitivity of SAMPEI for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI is implemented as a Python package, and is available from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI). The dataset is divided in 3 set of files: 1. Agnostic_discovery_benchmarking.zip file contains analyses performed to establish relative sensitivity and specificity of agnostic PTM discovery (Figure 2, Figure S3). 2. Chemoproteomics_of_LPS_stimulated_macrophages.zip file contains RAW264.7 cell proteomics identification results from X!tandem and SAMPEI (Figure 3, Figures S4-S7). 3. Itaconate_adducts_validation.zip file contains analysis to confirm cystein adducts produced by itaconic acid (Figure 5, Figures S8-S12).