Heavy arginine incorporation into newly synthesized histones before and after blocking HeLa cells in S-phase using thymidine. (A) Cells were grown in heavy media for 24 h or one doubling so that the ratio of new-to-old histone (heavy-to-light) should be 1:1. They were then blocked with thymidine. Time points were collected, and histones were analyzed using bottom-up MS. (B) MS1 spectra of H2B1C/2E/1 J/1 K(1–29), H2B1D(1–29), and H2A.Z(35–40) showing increase in heavy-labeled ‘new’ protein over time. H2B1D(1–29) and H2B1C(1–29) synthesis is blocked, and so, the proteins stopped incorporating heavy arginine after the double-thymidine block at 24 h. The synthesis of replication-independent H2A.Z continued after the double-thymidine block. (C) Plot of heavy arginine incorporation over time for replication-dependent and replication-independent histone isoforms. H2A.Z (blue) is a known replication-independent isoform, and H2A2B is a replication-dependent isoform (red). The replication-independent H2A.Z isoform continues to incorporate heavy arginine following the thymidine block, but replication-dependent histones stop incorporating the label.