Chemical derivatization of histones for LC-MS/MS analysis

ABSTRACT: Histone lysine methylation is an important post-translational modifications (PTMs) that plays critical roles in numerous biological processes with abnormal histone lysine methylation having been associated with developmental defects and human diseases. The primary amine of all lysine residues can be mono-, di-, or trimethylated and the extent of methylation at a single site is shown to inspire unique protein function. Moreover, histone lysine methylation can activate or repress transcription depending on which sites are modified and to what degree. Therefore, a comprehensive stoichiometric analysis of histone lysine methylation is necessary to fully elucidate its function. As histones are lysine-rich and highly-hydrophilic proteins (Figure S1), trypsin digestion of histones results in small, hydrophilic peptides which often suffer from substantial losses during sample preparation, and are difficult to detect in conventional reversed phase LC-MS. Additionally, trypsin fails to cleave histone proteins when lysine residues are methylated, resulting in inaccurate estimations of site occupancy and methylation extent. Previously, lysine propionylation labeling has been developed to overcome this drawback and facilitate quantitative analysis of PTMs that frequently occur on the lysine residue of histones. However, propionylation labeling of unmodified and monomethylated lysine residues causes a mismatch in hydrophobicity and charge state between labeled and unlabeled di-/trimethylated peptides. This mismatch forces retention time and signal intensity differences that inspire inaccurate quantitation and miscalculation of site occupancy. In this work, we developed a method that facilitates blocking of free lysine groups and discrimination of native lysine methylation, enables accurate calculation of the histone lysine methylation stoichiometry.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Junfeng Huang  

LAB HEAD: Lingjun Li

PROVIDER: PXD028269 | Pride | 2022-04-04

REPOSITORIES: Pride