Update information. Protein methylation plays important roles in DNA damage signaling. To date, there is still a lack of global profiling of whole-cell methylation changes during the DNA damage response and repair. In this study, using HILIC affinity enrichment combined with MS analysis, we conducted a quantitative analysis of the methylated proteins in HEK293T cells in response to IR-induced DNA damage. In total, 235 distinct methylation sites responding to IR treatment were identified, and 38% of them were previously unknown. Multiple RNA-binding proteins were differentially methylated upon DNA damage stress. Furthermore, we identified 14 novel methylations in DNA damage response-related proteins. Moreover, we validated the function of PARP1 K23 methylation in repairing IR-induced DNA lesions.