Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Abstract

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

Fig. 1.

Method for analysis of histone H4 N-terminal tail (residues 1–23) combinational codes. (A) Display of the nHPLC chromatogram and the associated N-terminal peptides. (B) Demonstration of the high mass accuracy mass spectra resulting from the pentaacetylated peak. This low-level peak displays four different isoforms: non-, mono-, di-, and trimethylated versions. (C) Depiction of ETD-MS/MS analysis of the dimethylated, pentaacetylated species. This spectrum reveals that K5, K8, K12, K16, and the N terminus are acetylated but K20 is dimethylated in this peptide.

Fig. 2.

Map of the 74 histone H4 combinational codes detected in human ES cells (cell line H1). Bracketed isoforms are positional isomers that coeluted and the corresponding percentages indicate the amount of this whole set (i.e., the global isoform percentage, GP). Shown in the right-hand column (Isomer Quant) is the amount of each positional isomer for each of these subsets. For instance, four diacetylated tails were detected and these four forms constituted 10% of the H4 population. By use of ETD-MS/MS we assigned the percentages of the four forms as 17, 6, 11, and 63. This means that the H4 tail having both the N terminus and K16 acetylated constitutes ≈6.3% of the entire histone H4 population in control human ES cells (0 h). Also shown are the percentages of each form at the 15 and 30 h TPA treatment time points. NA indicates either that (i) that form was present in that particular sample at levels that were too low to acquire reliable ETD data or (ii), in the case of triacetylated forms, that the combinations of modifications make it mathematically impossible to quantify coeluting isomers. All triacetylated isoforms were sequenced manually.

Fig. 3.

Quantification of histone H4 isoforms. (A) Four synthetic H4 tails varying in the number of acetylation sites were prepared, mixed in known ratios, and analyzed by MS. These data show that a linear response that matches closely with the theoretical (solid line). Three technical replicates of each mixture were performed resulting in three observed GPs for each expected GP. (B) Positional isomer quantification was validated by use of two synthetic tails having the same number of modifications, but only varying in placement. Here, the c- and z-type ion fragments generated by ETD are used to localize and quantify the relative amounts of each. Each data point represents the average of three or four consecutive ion ratios. Although there is a certain amount of systematic error as the ratio of K16Ac to K5Ac is increased, a good linearity is observed as the ratios are varied.