Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS

Abstract

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.

Keywords: FT-ICR; FTMS; histone; post-translational modification; proteoform; sequence variants.

Figure 1.

Sequence variants of human HeLa histone H2A (top) and H2B (bottom). (www.uniprot.org) Variants in bold have been previously identified in HeLa cells. (www.uniprot.org)

Figure 2.

Work flow for the identification and relative quantitation of histone H2B proteoforms from HeLa cells. A) Intact protein mass spectral segment for the 17+ charge state. Each isotopic distribution may present more than one H2B proteoform. B) Precursor ions of interest were isolated by an external quadrupole mass filter followed by in-cell SWIFT mass-selective excitation to within a nominal m/z range of ~1. C) Representative electron capture dissociation (ECD) product ion spectrum. Fragments are assigned based on a custom-input proteoform database by our custom software without prior deconvolution. Proteoform identification was then confirmed manually. D) Relative abundance ratios for corresponding fragments of different proteoforms from a given isolated precursor. E) Partial results of relative quantitation of histone H2B proteoforms.

Figure 3.

ESI 9.4 T FT-ICR mass spectral segments from histone fractions H2A1, H2A2a, H2A2b, and H2B from asynchronous HeLa cells. Proteoforms identified by ECD MS/MS are labeled. Because H2A fraction 2a (containing 2 major proteoforms) and H2A fraction 2b (containing 5 major proteoforms) were not baseline-resolved by RP-HPLC, proteoform H2A1D Nα-ac and H2A2B Nα-ac are present in both fractions.

Figure 4.

A) Sequences and putative ECD fragments for H2B1C, H2B1J, and H2B1O. Because of very small mass differences (~2 Da), these species will be isolated and fragmented together. Segments in orange or purple represent regions for which ECD fragments are identical in mass for 2 proteoforms. These three species yield unique fragments (segments in light blue, dark blue, and gray) only in the region z87-z124. B) Mass spectral segment for the z90 10+ ions of H2B1C and H2B1J to show the resolving power required for performing reliable top-down ECD MS/MS. The mass difference between H2B1C-z90 and H2B1J-z90 is the difference between ¹⁶O and ¹²C¹H₂ (1.97926 Da) and the split at m/z 993.83 is due to the small mass difference between ¹⁶O¹²C₂and ¹³C₂¹²C¹H₂ (27.4 mDa). Note that the minimum required resolving power at m/z 1,000 is 360,000. C) Mass scale-expanded segment, showing the narrow split at m/z 993.83 between ¹⁶O¹²C₂ and ¹³C₂¹²C¹H₂ (27.4 mDa). CD) Abundance ratio for the c ions (orange crosses) or z ions (blue squares) generated from H2B1C, H2B1J, or H2B1O for the m/z 811 precursor from HeLa cells. The ratio for the c-ion and z-ion series spanning AA1 to AA38 represents the ratio between the sum of H2B1C and H2B1J to H2B1O, whereas those in the range of AA39 to AA125 reflect the ratio between H2B1J and the sum of H2B1C and H2B1O. From the expressions at bottom left, the relative abundances of H2B1C, H2B1J, and H2B1O were calculated to be 3.2:1.0:0.82, in agreement with the numbers derived from the resolved peaks in Figure 4B.

Figure 5.

Relative abundances of histones H2A (top) and H2B (bottom) variants for HeLa cells, human embryonic stem cell line (WA09), U937 cell line, and prostate cancer cell line LaZ.