Mass Spectrometry of Protein Post-Translational Modifications Protocol v1

This protocol describes harvesting whole-cell protein lysates in RIPA buffer, immunoprecipitating a target protein (e.g., FOXA1) using a specific antibody with Protein A magnetic beads alongside an IgG control, and validating target enrichment by Western blot using input, IP, flowthrough, and control fractions. Enriched IP material is then resolved by SDS-PAGE, Coomassie-stained, excised around the target molecular weight, and prepared for LC–MS/MS to identify peptides and post-translational modifications, with database searching and FDR-controlled peptide filtering. Expected results include robust enrichment of the target protein in the specific IP compared with IgG and flowthrough, confirming successful pulldown prior to mass spectrometry. LC–MS/MS analysis should yield high-confidence identification of target-derived peptides and associated PTMs (e.g., phosphorylation, acetylation, ubiquitinylation), with low false-discovery rates and minimal background contributions from gel-only controls.