Method used for quantitative mapping of SUMO sites. (A) Schematic representation of the approach. (B) Theoretical MS spectra expected for peptides conjugated to SUMO or non-SUMO modifiers and to peptides over-SUMOylated or deSUMOylated in condition #2 versus condition #1. rel. ab., relative abundance, (C) MS spectra obtained for two different GG-modified peptides. The peptide from polyubiquitin-B is present in its light-labeled form and therefore was considered a contaminant, most likely derived from K63-linked polyubiquitin chains. The peptide from GTF2I (General transcription factor II-I) displays a SUMO1 site on K221. The absence of the peptide in its light-labeled form excludes GG modification by a non-SUMO modifier (i.e., ubiquitin, Nedd8, or ISG15). The decrease in intensity of the heavy-labeled form of this peptide indicates a decrease in the SUMOylation level of this site after LLO treatment. (D) Overlap between SUMO1 and SUMO2 sites identified in this study.