Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS.
Rijing LiaoDan ZhengAiying NieShaolian ZhouHaibing DengYuan GaoPengyuan YangYanyan YuLin TanWei QiJiaxi WuEn LiWei YiPublished in: Journal of proteome research (2017)
Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.
Keyphrases
- liquid chromatography
- mass spectrometry
- tandem mass spectrometry
- high resolution mass spectrometry
- ms ms
- dna methylation
- high performance liquid chromatography
- simultaneous determination
- ultra high performance liquid chromatography
- gas chromatography
- solid phase extraction
- liquid chromatography tandem mass spectrometry
- electron transfer
- capillary electrophoresis
- gene expression
- induced apoptosis
- healthcare
- social media
- emergency department
- multiple sclerosis
- physical activity
- single cell
- health information
- gas chromatography mass spectrometry
- oxidative stress
- drug induced