Unambiguous Determination of Isobaric Histone Modifications by Reversed-Phase Retention Time and High-Mass Accuracy

Methylation and acetylation of lysines are crucial posttranslational modifications that regulate gene tran- scription and have been shown to be misregulated in many forms of cancers. Western blot, immunopre- cipitation, and immunofluorescence are commonly used to characterize histone acetylation and methylation. However, these approaches are limited by the availability, site specificity, and cross-reactiv- ity of antibodies. Mass spectrometry is emerging as an additional powerful tool for histone characteriza- tion. The isobaric nature of trimethylation and acetylation (42.0470 and 42.0106 Da, respectively) confounds histone characterization by means other than high-resolution/high-mass accuracy mass spec- trometry. In this study, we adapted methodology that exploits difference in the relative retention time of acetylated and methylated peptides to unequivocally distinguish between these two modifications even with low-mass accuracy mass spectrometers. The approach was tested on tryptic digest of Saccharomyces cerevisiae histones. We found that acetylation resulted in increased retention in reversed-phase chroma- tography, whereas methylation, including trimethylation, showed little change in retention. For example, the acetylated forms of peptide 27