Purification and Analysis of ADP-Ribosylated Peptides

Products of ADP-ribosylation reactions containing 10 μg of protein were purified using standard TCA precipitation and resuspended in PBA buffer (100 mM HEPES pH 8.5, 150 mM NaCl, 2 mM MgCl₂). If required, cysteines were reduced in 10 mM dithiotreitol for 30 min at room temperature and alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark. Proteins were digested with 100 ng trypsin (Promega) overnight at 37 °C and peptides bound to m-aminophenylboronic acid agarose beads (Sigma) for 1 h at 4 °C. The beads were washed extensively with PBA buffer and the ADP-ribosylated peptides eluted with 1 M hydroxylamine (Sigma) pH 7.0 overnight at room temperature. Eluates were purified using C₁₈ ZipTips (Millipore) according to manufacturer instructions and analysed by nano-LC-MS (ThermoFisher U3000 nanoLC and Orbitrap XL mass spectrometer)39 (link). The raw mass spectrometry and tandem mass spectra were converted to.mgf format using Compass39 (link) and searched against the SwissProt database using Mascot (Matrix Science). Search parameters employed a precursor tolerance of 7 p.p.m. and a fragment ion tolerance of 0.8 Da. Quantification of precursor ions employed Skyline40 (link). ADP-ribosylation sites were identified based on a characteristic +15.0109 Da shift on glutamate and aspartate residues41 (link).