Anti-Flag immunoprecipitated 3×Flag-SUMO-T86R proteins on beads were washed once and suspended in 50 mM ammonium bicarbonate, followed by the addition of 1 µg trypsin/Lys-C mix and an overnight incubation at 37ºC. The digests were reduced, alkylated, diluted 10-fold with HBS (50 mM HEPES-NaOH pH7.5, 150 mM NaCl), and incubated with PTMScan HS K-ε-GG Remnant Magnetic Immunoaffinity Beads (Cell Signaling Technology) for 2 h at 4ºC. After washing with HBS three times, peptides were eluted with 200 µL of 0.1% TFA and 60% ACN. The eluates were evaporated, dissolved in 0.1% TFA, and desalted with GL-Tip SDB. After evaporation, the resultant peptides were dissolved in 0.1% TFA and 3% ACN and analyzed by an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer. The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column with a linear 4-32% ACN gradient for 0-60 min followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 seconds. Raw data were directly analyzed against the UniProt database restricted to Drosophila melanogaster using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to 2 missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) acetylation of protein N-terminus, oxidation of methionine, carbamidomethylation or N-ethylmaleimidation of cysteine, and di-glycine modification of lysine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.