Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry

Abstract

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

FIGURE 1.

PTM-dependent fractionation of histone H4 by HILIC enables detection of minor multiply modified forms. RP-H4 prepared from asynchronously growing HeLa S3 cells was fractionated on a Poly CAT A column essentially as described previously (25). A, this chromatogram is representative of three HILIC experiments performed. Top Down MS analysis of every fraction throughout the elution volume of H4 forms revealed that they generally eluted in order of increasing hydrophilicity with relative retention inversely related to their degree of ε-N-acetylation and Lys-20 methylation. The entire complement of H4 forms characterized and quantitated after HILIC fractionation is listed in Table 1. B, TDMS of fractions 36–57 show multiple intact masses corresponding to 0, 1, or 2 internal acetylations and 0, 1, 2, or 3 methylations.

FIGURE 2.

RP-HPLC followed by HILIC allows a >10⁴ dynamic range for detection of H4 forms. A, fraction 41 contains ∼0.07% of the total observed histone H4 area in the HILIC chromatogram (Fig. 1). B, FTMS of fraction 41 from A shows the presence of at least three different intact masses. C, ECD of the isolated mass 11387 Da in B identifies at least two isomeric forms present at ∼50% each. Using the MS and chromatographic signal levels, a dynamic range of ∼7 × 10⁴ is possible, translating to the ability to detect a H4 form present in ∼500 nucleosomes of the ∼32 million total per cell. S/N, signal-to-noise ratio.

FIGURE 3.

The relationship between acetylation site occupancies and the methylation state of Lys-20 for three acetylation states of H4 in asynchronous cells, monoacetylated H4 (A), diacetylated H4 (B), and triacetylated H4 (C). These results are representative from three separate HILIC experiments.

FIGURE 4.

Characterization of novel forms of histone H4. A, a HILIC chromatogram of H4 from the G₁/S border (0 h) with fractions containing novel H4 PTMs shaded dark gray. The corresponding MS of the 12+ charge state are shown in B and D, respectively. B, a monoisotopic mass of 11359 Da (corresponds to a Δm of +98 Da, shaded gray) was subsequently isolated and fragmented by ECD. C, the MS/MS data show that a Δm of +70 Da on fragment ions less than c₂₀ correspond to an N-terminal acetylation (+42) and two methylations (+28), with the latter localized to Arg-3. Fragment ions containing the first 20 residues or more were found to have a Δm of +98 Da, localizing +28 Da to a dimethlyated Lys-20. D, a monoisotopic mass of 11,289 Da (shaded gray) corresponds to a Δm of 28 Da, which is equivalent to 2 methylations. E, in a similar fashion to C, all fragments before Lys-20 showed no modification, whereas fragments after Lys-20 exhibit a +28 Da shift. If an N-terminal acetylation was present, fragments before Lys-20 would exhibit a +42 Da mass shift.

FIGURE 5.

Representative HILIC chromatograms for each of the following cell cycle time points: 0, 8, and 14 h. These time points correspond to the G₁/S border, late S/G₂, and mid-G₁ of the cell cycle, respectively. A, because all histone H4 is co-translationally acetylated at aαSer-1 (54), a form that is acetylated only at the N terminus and lacks any internal acetylations, is referred to as unacetylated (0Ac). Labeled here are the diacetylated (2Ac), monoacetylated (1Ac), and unacetylated (0Ac) regions. The unacetylated and monoacetylated regions are shaded to delineate fraction collection boundaries every minute. Alternately shaded regions reflect sample collection boundaries. Graphical representation of global abundance of unacetylated (B) and monoacetylated (C) of total H4 by methylation state at lysine 20 (0mLys-20, 1mLys-20, and 2mLys-20) is shown. D, the relationship between relative monoacetylation site occupancies of aLys-5 (•), aLys-8 (▴), aLys-12 (▪), and aLys-16 (♦) to the methylation state of Lys-20 during the cell cycle.