Examining histone posttranslational modification patterns by high-resolution mass spectrometry

Abstract

Histone variants and posttranslational modifications (PTMs) are essential for epigenetic regulation of transcriptional expression. Single and/or combinatorial PTMs of histones play important roles in development and disease formation. Mass spectrometry (MS) has been a powerful tool to study histone variants and PTMs, as it not only can identify novel PTMs but also can provide quantitative measurement of a spectrum of histone variants and PTMs in the same sample. In this chapter, we employ a combination of chemical derivation and high-resolution MS to identify and quantify multiple histone variants and PTMs. Histones are acid extracted and modified with propionyl groups and subsequently produces suitable sizes of fragments for MS analysis by trypsin digestion. The newly generated N-termini of histone peptides can be differentially marked by stable isotope labeling in a second reaction of propionylation, which enables direct comparison between two different samples in the following MS analysis.

Figure 1

Offline RP-HPLC separation of histones. H1, H2B, H2A1, H4, H2A2, H3.2, H3.3 and H3.1 are eluted in a time-wise manner.

Figure 2

Chemical derivation of histones. An example peptide with an N-terminal lysine residue (boxed) and an intermediated arginine residue (boxed) is shown. R1 and R2 represent other amino acid residues. After the propionylation treatments before trypsin digestion, the N-terminal amine group (in blue) and the free amine group (in blue) on the lysine residue are both modified with a propionyl group (in red). Propionic acids are the side products of this reaction. Trypsin cleavages at the C-terminal of the arginine residue, which produces two shorter peptides. Propionylation after the trypsin digestion adds propionyl group (in red) to the newly generated N-terminus (in blue).

Figure 3

Set up of stage-tip and TiO₂ micro-column. A. Stage-tip: the C18 mini disks are transferred to a P200 tip using a P1000 tip. A fused silica was used to push the C18 mini disks down to the bottom of the p200 tip. The arrow shows the C18 plug after all C18 mini disks are securely wedged. B. TiO₂ micro-column: the bottom layer of C8 plug turned clear after activation by methanol and the top TiO₂-bead layer looked white. 1.5 mg of TiO₂ was used to make this micro-column.

Figure 4

MS chromatogram of the [M+2H]²⁺ ions of histone H3 9–17 peptide. The peptides are propionylated and digested by trypsin. The modifications and m/z values are indicated. The peptides are identified by their m/z and MS/MS spectrum. The area under each peak represents the abundance of each ion and can be measured in the Qual Browser.

Figure 5

MS/MS spectrum of two [M+2H]²⁺ precursor ions from the digest of propionylated histone. Both peptides were determined to span histone H3 residues 9 to 17. A. the peptide is unmodified; B. the peptide is mono-methylated on K9 and acetylated on K14. All b and y ions are found and labeled. Inset: mass spectrum of the precursor parent ions. Calc. calculated m/z; expt. experimental m/z.

Figure 6

MS chromatogram of three charge states of a histone H3 9–17 peptide with K9 or K14 acetylated. The area underneath each peak is measured either by automatic area (AA) or manual area (MA) measurement. BP represents the base peak m/z.

Figure 7

MS/MS spectrum of the [M+2H]²⁺ precursor ions in Fig. 4E, representing two histone H3 9–17 peptides with one lysine residue (K9 or K14) to be acetylated. Most b and y ions are found and the most abundant ones are labeled. The ions from the K9ac peptide are in red; and the ions from the K14ac peptide are in blue; the ions shared between the two peptides are in black. In this particular experiment, the majority of signal comes from the K14ac peptide.

Figure 8

Differential expression MS analysis of histone PTMs. A and B. MS chromatogram of the histone H3 41–49 peptides from sample 1 and 2, which were labeled with D0 and D10 propionic anhydride in Step 6.3, respectively. The propionyl groups that were added to the N-termini of peptides contain 5 hydrogen (D0) or 5 deuterium (D5) atoms (Fig. 2). C and D. MS chromatogram of the histone H3 9–17 peptides with K9me1 from sample 1 (D0) and 2 (D5), respectively. AA, automatic area measurement; BP: base peak m/z, z=2.