Middle‐Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms

Abstract

Post-translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle-down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) for on-line liquid chromatography-mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to unambiguously distinguish between acetylation and tri-methylation (m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono-and di-methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle-dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

“…However, each of the four histone proteins can be post-translationally modified at multiple locations, and with several different chemical marks 32 . Proteomic studies have demonstrated that each histone tail caries a unique set of modifications 33 , and that the abundance and availability of specific histone proteoforms is dynamic 34,35 . The BET bromodomains BRD2, BRD4 and BRDT are all able to bind diacetyllysine modifications including H4K5acK8ac, H4K5acK12ac and H4K12acK16ac 4,36 .…”

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…83 Additionally, in a very recently published study, phosphorylation of histones (H4) have been identied even in breast cancer cells (MDA-MB-231 and MCF-10A) at different cell cycle stages. 266 Furthermore, tissue-specic variations in PTMs of histones H3 in a rat model was reported by Garcia et al, for which tissues from kidney, spleen, brain, bladder, lung, liver, heart, ovary, testes and pancreas were analyzed by MD proteomic approach. 73 PTMs on histones have also been investigated and quantitated in mouse embryonic stem cells by MD based workow, which involved the use of protease GluC and WCX-HILIC chromatography coupled online to ETD MS/MS.…”

Current Trends in the Analysis of Post-translational Modifications

“…Acetylated residues, therefore, can be separated from their non-acetylated counterparts based on hydrophobicity (RP-HPLC), electrostatic interactions (SCX), or both (zwitterionic hydrophilic interaction liquid chromatography, ZIC-HILIC) [64,68]. HILIC also has the added value of being able to separate methylated and acetylated histones, significantly decreasing sample complexity [69]. Another possibility for reducing sample complexity is derivatization by propionic anhydride, which converts lysine residues and the N-terminal amino group into propionyl amides providing a + 56 Da mass shift and protection from enzymatic digestion [37,70].…”