There are ~70 protein methyltransferases (PMTs) encoded in the human genome, but over 16,000 characterized methylation events occurring on histone and nonhistone substrates. This suggests that each PMT has both a canonical function and noncanonical activities contributing to its biological role(s). Since interrogating protein methylation poses challenges in the forms of redundancy, transiency, and specificity, these noncanonical activities are still largely undiscovered. Our lab has developed a technique called Bioorthogonal Profiling of Protein Methylation (BPPM) that has lifted the curtain on interrogating PMT activities. Applying this technique to two PMTs, DOT1L and NSD2, we have uncovered a novel histone target for each enzyme. For DOT1L, BPPM has revealed the substrate H4K5. Further interrogation of this activity has shown that this activity is specific to MLLr leukemia, where it is read by the transcriptional coactivator SPIN1. Dependent on DOT1L activity, SPIN1 binds and induces expression of key MLLr genes, shown by CUT&TAG and RNAseq. Furthermore, SPIN1 shRNA knockdown causes growth impairment in MLLr cells. This indicates DOT1L-catalyzed noncanonical H4K5 methylation contributes to MLLr cell proliferation through SPIN1. In another study, we were interested to interrogate NSD2’s role in the DNA damage response after a recent paper had debunked its proposed activity of H4K20me2 to recruit 53BP1. Hypothesizing that another histone mark may be involved, we used BPPM to discover NSD2-mediated methylation of H3K18. While we could confirm loss of NSD2 impairs DNA repair by I-SceI-based assays and quantification of 53BP1 foci, we could not measure the role of H3K18me1 likely due to PMT redundancy. Therefore, further investigation is necessary to study the role of H3K18 methylation in the recruitment of 53BP1.