diGLY Immunoprecipitation for TMT Proteomics

diGLY capture was performed largely as described (25 (link)). The diGLY monoclonal antibody (Cell Signaling Technology; D4A7 clone) (32 μg of antibody/1 mg of peptide) was coupled to Protein A Plus Ultralink resin (1:1 μl of slurry/μg of antibody) (Thermo Fisher Scientific) overnight at 4°C before its chemical cross-linking reaction. Dried peptides (1-mg starting material) were resuspended in 1.5 ml of ice-cold immuno affinity purification (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 hours at 4°C with gentle end-over-end rotation. After centrifugation at 215g 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% trifluoroacetic acid, desalted using homemade StageTips, and dried via vacuum centrifugation, before TMT labeling. A detailed protocol describing the diGLY immunoprecipitation for TMT proteomics has been reported (dx.doi.org/10.17504/protocols.io.buyunxww).