Generation of SUMO variants compatible with quantitative GG-capture proteomics. (A) Comparison of C-terminal sequences in mature SUMO1, SUMO2, ubiquitin, Nedd8, and ISG15. C-terminal GG motifs are highlighted in purple. The positions of mutations introduced in SUMO1 and SUMO2 are indicated in green. (B) Schematic representation of the signature tags left after trypsin digestion on peptides modified by either wild-type or T95R SUMO1. (C) Comparison of the patterns of SUMO-conjugated protein in cells expressing wild-type or variant SUMOs. SUMOylated proteins from HeLa cells transfected with wild-type or variant His₆-SUMOs were pulled down and analyzed by immunoblot using anti-SUMO1 or anti-SUMO2/3 antibodies. (D) Detection of RanGAP1 SUMO-conjugated forms by immunoblot analysis of HeLa cells expressing wild-type or variant His₆-SUMOs. Input fractions are shown as control.

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.

Validation of identified SUMOylated proteins and SUMO sites. HeLa cells were cotransfected with His₆-SUMO1 or -SUMO2 and with HA-tagged wild-type or K-to-R mutant expression vectors for selected candidates. Cell lysates were subjected to His pulldown (His PD), and the presence of SUMOylated forms of the different candidates was assayed using anti-HA antibodies. Input fractions are shown as control.

Analysis of identified SUMO1- and SUMO2-conjugation sites. (A) GO terms enrichment analysis of proteins conjugated to SUMO1 or SUMO2 compared with all human proteins. Bars correspond to the percentage of proteins annotated with each GO term. Asterisks indicate groups with significant enrichment (modified Fisher exact P value < 0.0001). (B) Distribution of SUMO1 and SUMO2 sites over different types of SUMOylation motifs. (C) IceLogo () representations showing the amino acids surrounding the SUMO-conjugated lysine residue in different motifs. Imposed amino acids for each type of motif are marked by an asterisk. The frequency of nonimposed amino acids at every subsite is compared with sampled frequencies in the human proteins stored in the UniProt/Swiss-Prot database (negative control). Only residues that are statistically overrepresented (upper part of the iceLogo) or underrepresented (lower part of the iceLogo) at a 95% confidence level are depicted. Residues that never were observed at specific positions are shown in pink.

Analysis of targets deSUMOylated in response to the LLO toxin. (A) Immunoblot analysis of SUMO-conjugated patterns from cells transfected with His₆-SUMOs variants and exposed to LLO. (B) Functional enrichment analysis of highly deSUMOylated proteins after LLO treatment compared with all SUMO-conjugated proteins. Bars correspond to the percentage of proteins annotated with each Splice Pattern-Protein Information Resources (SP-PIR) keyword. Asterisks indicate groups with significant enrichment (modified Fisher exact P value < 0.05).