Immunoaffinity Enrichment of diGLY Peptides

diGLY capture was performed largely as described (Rose et al., 2016 (link)) . The diGly monoclonal antibody (Cell Signaling Technology; D4A7 clone) (32 μg antibody/1 mg peptide) was coupled to Protein A Plus Ultralink resin (1:1 μL slurry/ μg antibody) (Thermo Fisher Scientific) overnight at 4°C prior to its chemical cross-linking reaction. Dried peptides (indicated amount in corresponding figures) were resuspended in 1.5 mL of ice-cold IAP buffer [50 mM MOPS (pH 7.2), 10 mM sodium phosphate and 50 mM NaCl] and centrifuged at maximum speed for 5 min at 4°C to remove any insoluble material. Supernatants (pH ∼7.2) were incubated with the antibody beads for 2 hr at 4°C with gentle end-over-end rotation. After centrifugation at 215 × g for 2 min, beads were washed three more times with ice-cold IAP buffer and twice with ice-cold PBS. The diGLY peptides were eluted twice with 0.15% TFA, desalted using homemade StageTips and dried via vacuum centrifugation, prior to TMT labeling.