Yang L, et al. (2010) Unambiguous determination of isobaric histone modifications by reversed-phase retention time and high-mass accuracy. Anal Biochem 396(1):13-22
Abstract: Methylation and acetylation of lysines are crucial posttranslational modifications that regulate gene transcription and have been shown to be misregulated in many forms of cancers. Western blot, immunoprecipitation, and immunofluorescence are commonly used to characterize histone acetylation and methylation. However, these approaches are limited by the availability, site specificity, and cross-reactivity of antibodies. Mass spectrometry is emerging as an additional powerful tool for histone characterization. The isobaric nature of trimethylation and acetylation (TriMe = 42.0470 Da, Ac = 42.0106 Da) confounds histone characterization by means other than high-resolution / high-mass accuracy mass spectrometry. In this paper we adapted methodology that exploits difference in the relative retention time of acetylated and methylated peptides to unequivocally distinguish these two modifications even with low-mass accuracy mass spectrometers. The approach was tested on tryptic digest of S. cerevisiae histones. We found that acetylation resulted in increased retention in reversed-phase chromatography, while methylation, including trimethylation, showed little change in retention. For example, the acetylated forms of peptide (27)KSAPSTGGVKKPHR(40) eluted at 15.63 min whereas the methylated forms eluted at 13.89 min. In addition, the effect of acetylation was cumulative as observed in the case of peptide (9)KSTGGKAPR(17), whose un-, mono-, and diacetylated isoforms eluted at 7.43, 10.47, and 16.49 min, respectively. The modification patterns of the peptides in question were subsequently verified by high-mass accuracy tandem mass spectrometry.
|Status: Published||Type: Journal Article||PubMed ID: 19699711|
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