Research Paper Volume 4, Issue 11 pp 823—842

Figure 5. Relative abundance of unique phosphorylated, unphosphorylated C-terminal, and internal monitoring H2A.X peptides measured by SRM and qPCR.

(A) H2A.X sequence indicating tryptic peptide sequences common to all H2A family members (blue) and unique to H2A.X (red). The location of serine phosphorylation found in γH2A.X is marked. (B) qPCR analysis of H2A.X mRNA expression in proliferating, acute DNA damage, and drug-evoked senescence HCA2 cells, as described in 3B. Biological and technical triplicates were performed for each condition. The p-values were calculated using Student's t-test, two-tailed, unequal variance. (C) Relative nuclear abundance of the unique internal monitoring H2A.X peptide, TSATVGPK, as measured by SRM. Eight biological replicates were used for each condition. (D, E). Relative nuclear abundance of the unique C-terminal phosphorylated and unphosphorylated H2A.X peptides. The fully tryptic ATQASQEY (D) and one missed cleavage KATQASQEY (E) peptides and their phospho-Ser-139 analogs (γH2A.X) were quantified using SRM. Eight biological replicates were used for each condition, for each peptide.