Summary of overrepresented motifs at PTM sites.

<p>A total of ten motifs were detected, flanking either acetylation, methylation or phosphorylation sites. For each motif, bibliographic references for the same or similar motifs are listed, as well as whether it is known to function as a binding motif. Unknown motifs were novel at the time of writing. Many of the identified motifs are novel and distinct from human motifs in the human protein reference database (HPRD). In support of our dataset many of the sites also matched known motifs for the enzymes that catalyse these PTMs, and/or known binding motifs that require modified residues.</p

A,B) Mass spectra of identified endogenous peptides with lysine propionylation and butyrylation and their synthetic counterparts.

<p>Major peaks are labelled in the mass spectra and the fragment ions indicated in the peptide sequence using standard Mascot nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036980#pone.0036980-Roepstorff1" target="_blank">[56]</a>. <b>A</b>) A novel site of lysine butyrylation on residue K95 of H2A. <b>B</b>) A novel site of lysine propionylation on residue K95 of H2A. <b>C</b>) Highly modified peptides that were detected using ETD-MS/MS included the N-terminal peptide from H4 ac-SGRGKacGGKacGLGKacGGAKacRHRKme2VLR, which contains 5 sites of acetylation and 1 site of dimethylation <b>E</b>) A novel site of lysine crotonylation on residue K108 of H2B.</p

Proportion of detected histone peptides in the adult mouse brain.

<p><b>A</b>) Workflow for the isolation and analysis of long histone peptides from the mouse brain. <b>B</b>) Number of peptides identified for each histone subtype, in brackets the number of unique peptides identified and typical sequence coverage observed.</p

All novel histone PTMs.

<p>Summary of all novel PTMs identified on H1 (<b>A</b>), H2A (<b>B</b>), H2B (<b>C</b>), H3 (<b>D</b>) <b>and H4</b> (<b>E</b>). Sites of PTMs are indicated by A for acetylation, B for butyrylation, Cr for crotonylation, Me1, Me2 and Me3 for mono-, di- and trimethylation, P for phosphorylation and Pr for propionylation. Residues are numbered starting with the first residue after the cleaved methionine. Canonical H1, H2A, H2B and H3 histones are shown which represent sequences common across all subtypes.</p

Combinatorial patterns on H2A and H2B.

<p>Summary of N-terminal H2B and H2A combinatorial codes identified using ETD-MS. Each line represents an individual H2B_1–25 (<b>A</b>) or H2A_1–41 (<b>C</b>) peptide. Probability of co-occurrence of individual PTMs on H2B_1–25 determined by an association rule data-mining algorithm (<b>B</b>). The condition (left rows) is when a specific PTM is observed on H2A/H2B, and the outcome (top columns) is the probability (indicated by a heat plot) that a second or several PTM(s) are observed at the same time on the same histone molecule. Diagram depicting the relationship between three PTMs on H2A_1–41 (<b>D</b>). N-term Ac, K5ac and R3Me3 were either mutually exclusive or always seen in combination. The frequency of each PTM is indicated by the % above the circle, connections indicate the derived rule and its % occurrence. For instance, when K5ac was present, N-term ac was also observed in 100% of cases (connected by arrow), but R3me3 was never observed. When R3me3 was observed, N-term acetylation or K5ac was never observed (100% of cases), suggesting mutual exclusion of N-term acetylation and R3me3, potentially due to steric hindrance or conformational changes induced by each PTM.</p

Summary of PTM abundance.

<p>The site, type and level of modification site occupancy of each PTM is indicated for each histone. For each PTM, the level of modification site occupancy is calculated by dividing the number of instances that a PTM was detected by the number of instances that a given amino acid was observed, providing an estimation of the relative abundance of each PTM. Sites of phosphorylation, which were detected only in IMAC/TiO₂ enriched fractions, are not listed. Residues are numbered starting with the first residue after the cleaved methionine. Canonical H1 (<b>1A</b>), H2A (<b>1B</b>), H2B (<b>1C</b>), <b>H3</b> (<b>1D</b>), and H4 (<b>1E</b>) histones are shown which represent sequences common across all subtypes.</p

Multiple PTMs occur in distinct combinations on H4.

<p><b>A</b>) Table depicting each of the 304 combinatorial codes identified on H4_1–24 by ETD-MS. The number of each residue carrying a PTM is indicated at the top and each line represents an individual peptide. Probability of co-occurrence of (<b>B</b>) individual PTMs and (<b>C</b>) individual PTMs with groups of PTMs, on H4_1–23 determined by an association rule data-mining algorithm. The condition (left rows) is when a specific PTM is observed on H4, and the outcome (top columns) is the probability (indicated by a heat plot) that a second or several PTM(s) are observed at the same time on the same histone molecule.</p