FIGURE 4.
Accrual of asparagine hydroxylation over time. A, extracted ion chromatograms of m/z 772.71 and 778.02 corresponding to unhydroxylated (dashed line) and hydroxylated (solid line) forms of the tryptic Rabankyrin-5 peptide encompassing Asn752; SGCDVNSPRQPGANGEGEEEAR ([M + 3H]3+). Nano-UPLC-MSE chromatography analysis demonstrating increase in the proportion of material that was hydroxylated over time in cells treated with cycloheximide (Chx) for 4 h (middle panel) and 10 h (lower panel). The signal intensity of reference peaks is indicated in parentheses on the chromatograms. B, panels i and ii, pulsed SILAC experiment demonstrating increase in the proportion of material that was hydroxylated at Asn485 and Asn797 in Rabankyrin-5 over an extended 48-h chase period in light media (Lys0/Arg0) following 24-h pulse labeling in media containing heavy isotopes (heavy media; Lys6/Arg6) (hydroxylation percentages shown for chase period only); nano-UPLC-MSE analysis. Two independent biological replicates are shown, and data presented are from one representative experiment.