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Review
. 2015 Mar 25;115(6):2376-418.
doi: 10.1021/cr500491u. Epub 2015 Feb 17.

Quantitative proteomic analysis of histone modifications

He Huang  1 , Shu Lin  2 , Benjamin A Garcia  2 , Yingming Zhao  1
Affiliations
Review

Quantitative proteomic analysis of histone modifications

He Huang et al. Chem Rev. .
No abstract available

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of histone post-translational modifications.
Figure 2
Figure 2
MS/MS for peptide sequencing and PTM detection. (A) Nomenclature for fragment ions in mass spectra for peptides (modified from ref 45). Schematic showing a five residue peptide. The vertical lines show the bond cleavage, and the horizontal lines show the paired-product ions formed. Red, a and x ions; green, b and y ions; blue, c and z ions. (B) A MS/MS spectrum example obtained by HCD. The N-terminal of this peptide and the lysine residue is propionylated, adding 56.026 Da. The parent ion is charge +2 with m/z equals to 408.732. The detected b and y ions are highlighted and labeled. (C) MS/MS spectra showing the same peptide that is monomethylated on the lysine residue, which adds 14.016 Da.
Figure 3
Figure 3
Schematic overview of the peptide fingerprint alignment.
Figure 4
Figure 4
An example of a mass shift caused by lysine acetylation. Insets show the precursor ion masses. In addition to the parent ions, the daughter ions containing the acetyllysine have also a mass shift of 42.0106 Da caused by acetylation.
Figure 5
Figure 5
Schematic showing SILAC followed by HPLC/MS/MS. Treatment and control cells are cultured in different media, containing “light” and “heavy” isotope-containing amino acids, respectively. Equal numbers of “light” and “heavy” cells are mixed for collecting proteins. The protein mixture is then digested by a protease of choice. The resulting peptide mixture can be subjected to HPLC/MS/MS analysis, with or without prior fractionation. The same peptide from the two cell populations can be identified and quantified by MS analysis.
Figure 6
Figure 6
(A) Chemical structures of ITRAQ, TMT, and DiLeu reagents, showing design principles for these reagents. In multiplexing reagents, the reporter group carries different numbers of 13C and/or 15N atoms, resulting one dalton different in mass among different tags. The balance group is also labeled by different numbers of stable isotopes. Thus, the combined report and balance groups have the same total molecular weights. The amine reactive group reacts with amine groups on peptides’ N-termini and unmodified lysine residues, adding the isobaric tag onto the peptides. (B) Example of multiplex proteomic quantitation with ITRAQ, TMT, or DiLeu reagents.
Figure 7
Figure 7
Bottom-up and top-down mass spectrometry methods. In the bottom-up method, proteins are digested by a protease such as trypsin, Arg-C, Glu-C, or Asp-N protease. The peptides are then subjected to HPLC/MS/MS analysis. The chromatogram shows the base-peaks of a bottom-up MS run. In the top-down approach, a purified protein is analyzed in MS that is either directly infused or separated in HPLC before MS analysis. ETD is usually chosen as the MS/MS fragmentation technique. The spectrum shows isotope distribution of a protein ion population of charge +8.
Figure 8
Figure 8
Progressive steps for identification, verification, and systematic analysis of novel PTMs in histones.
Figure 9
Figure 9
Verification of lysine 2-hydroxyisobutyrylation. (A) Extracted ion chromatograms (XICs) from HPLC/MS analysis of a mixture of four synthetic peptides (DAVTYTEHAK(±)-2ohbuR, DAVTYTEHAK(r)-3ohbuR, DAVTYTEHAK(s)-3ohibuR, and DAVTYTEHAK4ohbuR), without (top) or with (bottom) the in vivo peptide. The x axis represents retention time of HPLC/MS analysis, while the y axis represents the MS signal. (B) The MS/MS spectra of an in vivo peptide bearing a PTM (DAVTYTEHAK+86.0354R) (top), a synthetic lysine 2-hydroxyisobutyrylated peptide corresponding to the sequence of the in vivo peptide (middle), and a mixture of the two peptides (bottom). The label Δ designates b or y ions with water and/or ammonia loss. Insets show the precursor ion masses. The data are from the published literature.
Figure 10
Figure 10
Chemical derivatization of lysine-containing peptides by propionic anhydride. R1 and R2 represent amino acid side chains, and R3 represents other residues. After the derivatization, both the N-terminal amine group and the amine group on the unmodified lysine residue are modified with propionyl group. Propionic acids are the side products of this reaction.
Figure 11
Figure 11
HPLC/MS/MS analysis of the [M+2H]2+ ions of histone H3 9–17 peptide KSTGGKAPR. Eight forms with various PTMs were detected and shown. The peptides are propionylated and digested by trypsin. The PTMs and m/z values of the peptides are indicated.
Figure 12
Figure 12
Example of MS/MS achieved by ETD fragmentation for the H3 1–50 peptide with K23 acetylated (ac) and K27 dimethylated (me2). The raw spectrum was deconvoluted and deisotoped by the Xtract program as described., The relative abundance of the c4 ion (m/z = 474.313) was set to be 100%. Identified c and z ion peaks are annotated both on the peptide sequence and in the spectrum. The modified lysine residues and critical fragment ions, c23 and z24, are highlighted in red.
Figure 13
Figure 13
Averaged PTM data from 24 human cell lines; data achieved from ref 142). (A) Histone H3K4 PTMs; (B) H3K9 PTMs; (C) H3K27 PTMs; (D) H4K20 PTMs. The numbers above each bar indicate the percentiles of the particular PTM over the total H3 signal (100%). Error bars represent standard deviation among 24 cell lines.
Figure 14
Figure 14
Typical workflow of the proteomic analysis of PTMs.
Figure 15
Figure 15
Schematic representation of experimental workflow for quantitative proteomics of PTMs.
Figure 16
Figure 16
Schematic overview of the workflow for identifying histone-mark “binders”. A pair of biotinylated peptides are synthesized and used as baits to incubate with a protein lysate for pulling down experiment. Gray curves indicate immobilized histone tail peptide. Red triangle indicates histone mark. (Left) The proteins isolated with modified and unmodified peptides were resolved in SDS-PAGE; proteins specific to histone marks were visualized and then identified by MS. Alternatively, the enriched proteins from the two affinity enrichment experiments are digested and then analyzed by HPLC/MS/MS for identifying and quantifying proteins. The histone mark-specific binding proteins will be identified. (Right) SILAC-based quantitative proteomic approach for identifying and quantifying proteins that bind to a histone mark of interest.
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