RESEARCH ARTICLE Multiple Proteases to Localize Oxidation Sites

Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translation-al modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other pre-viously unidentified oxidative modifications. This work highlights the use of multiple prote-ase digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses