Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples

Abstract

Purpose Profiling histone posttranslational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. MS‐based approaches to analyze histone PTMs in clinical samples usually rely on SDS‐PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage. Experimental design and results The aim of this study is the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in‐solution digestion protocols. Among them, the Arg‐C digestion, which allows profiling histone H4 modifications, and the Prop‐PIC method, which improves the detection of short and hydrophilic peptides, are tested. This approach is validated on different types of samples, including formalin‐fixed paraffin‐embedded pathology tissues, and employed to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones. Conclusions and clinical relevance This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of histone marks in patient samples.

Profiling of Epigenetic Features in Clinical Samples Reveals Novel Widespread Changes in Cancer

“…Most of the changes observed in luminal A-like samples were also present in triple negative breast cancer samples, where additional changes could be observed (these included an increase of H3K9me3, H3K36me1, and H3K79ac, and a decrease of H3K27me3-containing peptides and H3K79me1/me2). This result is in agreement with what we have recently reported for histone H4 PTMs, where triple negative samples showed many more changes than luminal A-like samples, compared with normal tissues [18]. This finding is also consistent with the notion that luminal A-like samples have a lower proliferation rate, are less aggressive and typically more differentiated [19], and, as such, they may be more similar to normal tissues, compared with triple negative samples.…”

“…No change was detected at the levels of H3K27me3 or H3K36me1/me2, possibly because the cancer cell lines do not have a proliferation rate higher than the normal cells (the doubling times for MCF7, MDA-MB-231, and MCF10A are 41, 43, and 24 hours, respectively). Additionally, in luminal A cells, there was a decrease of the tetra-acetylated form of the histone H4 peptide 4–17, contrary to the increase that we have previously observed in luminal A-like tumor tissue [18]. The decrease of H3K14ac-containing peptides was lost in both breast cancer subtypes, while the increase of H3K9me3 was somewhat maintained (Figure 6B–D).…”

Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A—Treated Stem Cells

“…Unfortunately, the very high number of basic residues occurring within the small histone proteins leads to the formation of short peptides, impairing an effective MS analysis. Although alternative enzymatic digestion protocols have been tested [ 19 , 20 , 21 , 22 ], this drawback still remains challenging. Moreover, PTM heterogeneity generates isobaric peptides containing the same modification located at different positions along the peptide chain, making their localization very difficult [ 23 ].…”

Trimethylacetic Anhydride–Based Derivatization Facilitates Quantification of Histone Marks at the MS1 Level

“…However, some particular histone peptides (e.g., hydrophilic methylated H3K4 and long Arg-C-like N-terminal peptides of H2B variants) still pose challenges. For identifying methylated H3K4, Prop-PIC hybrid labeling seems to be the currently preferred method (26,(32)(33)(34)(35). This can reportedly shift retention times of monomethylated, dimethylated, and trimethylated forms of the T3-R8 peptide by 8 to 12 min (26).…”