Reductive Dimethylation Labeling of Peptides.

Cells were resuspended in 8 M urea lysis buffer and lysed by bead-beater disruption. Lysates were reduced with 4.5 mM DTT and alkylated with iodoacetamide. Proteins were digested with trypsin, and peptides were purified on C18 solid-phase extraction cartridges. Lyophilized peptides were labeled in vitro using reductive dimethyl labeling as previously described (59). For light cells (cells treated to induce DNA damage), peptides were dissolved in 1 M Hepes (pH 7.5) and were treated with 0.56 mL of 60 mM sodium cyanoborohydride and 0.56 mL of 4% formaldehyde for 20 min at room temperature. For heavy (untreated) cells, peptides in 1 M Hepes (pH 7.5) were treated with 0.56 mL of 60 mM (D3)-sodium cyanoborodeuteride and 0.56 mL of 4% deuterated (D2)-formaldehyde. Reactions were quenched with acetic acid, and dimethylated peptides were desalted using a Sep-Pak C18 cartridge (Waters). Light and heavy peptides were combined in a 1:1 ratio and lyophilized for further use.