Immunodetection of Cardiac Lysine Acetylation.

<p>Panel A. Guinea pig cardiac myofilaments were prepared essentially as described by Solaro <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Solaro1" target="_blank">[70]</a>. 30 µg of protein was subjected to electrophoresis and immunoblotting with both monoclonal and polyclonal antibodies to acetylated lysine as described in the Methods section. Lanes with immunoreactive bands are juxtaposed with the corresponding exposure of a parallel control experiment conducted with >2% w/v acetylated BSA in the primary antibody incubation step. Panels B and C. Macromolecular respiratory complexes were resolved as described in the methods. Up to 20 µg of protein from the 30% w/v sucrose fraction (enriched Complex I, Panel B) and the 22.5% w/v sucrose fraction (enriched complex V, Panel C) were probed for immunoreactivity to anti-acetylated lysine as described for Panel A.</p

Proteomic Work-flow.

<p>Asterisks denote that since the data from the microsomal and plasma membrane fraction yielded few new sites or proteins, it was combined with data from the cytosolic fraction for the purposes of discussion.</p

Lysine Acetylation of Two Key Proteins in EC Coupling.

<p>Panel A depicts a structural ribbon model of guinea pig SERCA2a with helices in cyan and beta strands in magenta; the membrane domain, the phosphorylation domain (P-domain) and the nucleotide binding domain (N-domain) are shown. Residues that are important for nucleotide binding are shown in salmon hue. The positions of lysine residues acetylated in our study (K⁴⁶⁴, K⁵¹⁰, K⁵³³) are confined to the distal end of the cytoplasmic nucleotide-binding domain (N-domain), and are highlighted in red. Panel B depicts a model of guinea pig myosin heavy chain beta S1 head. Helices and beta strands are shown in pale cyan for simplicity. The positions of lysines acetylated in the dataset are highlighted. Magenta residues represent acetylation sites identified by the most spectra. Red residues were identified by fewer spectra. Solid blue lysine residues are sites of acetylation that are mutated in HCM. Spotted blue residues indicate positions of HCM mutation immediately adjacent to an acetylation site. Brown residues denote the positions of lysine residues found to be acetylated in previous studies by Samant <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Samant1" target="_blank">[61]</a>. Lysines highlighted in the discussion are numbered for clarity in each panel.</p

The Dataset.

<p>Panel A. Distribution of acetylated proteins by experimental subfraction. Panel B. Distribution by biological replicate. Panel C. Comparison with published global-scale mammalian lysine acetylomes including: (*) mouse liver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Kim1" target="_blank">[4]</a>, (^&) human acute myeloid leukemia cell line, MV4-11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Choudhary1" target="_blank">[3]</a>, and (^%) human liver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Zhao1" target="_blank">[6]</a>. Panel D. Comparison with the (<a href="mailto:@" target="_blank">@</a>) mouse liver mitochondrial lysine acetylome of Schwer <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone.0067513-Schwer1" target="_blank">[35]</a>.</p

Functional Annotation and Enrichment analysis.

<p>Panel A. Distribution of acetylation sites by cellular component. Panel B. Distribution of acetylation sites by biological process. Data for panels A and B were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067513#pone-0067513-g003" target="_blank">Figure 3</a> and expressed as percentages of the total number of sites. Panel C. Network of gene ontology annotations for molecular function. Node color represents the magnitude of corrected p-value, a continuous variable that provides a measure of enrichment (see legend). Node size is proportional to the number of genes associated with each GO-term. The layout is broadly grouped by similar molecular functions, including catalytic activity, ligand binding and transport activity.</p

Top 40 Most Heavily Acetylated Proteins in the Heart (by Site Count).

<p>Top 40 Most Heavily Acetylated Proteins (by Number of Acetyl-Lysine Residues). Asterisks (*) designate the accessions of acetyl-proteins identified in all three biological replicates.</p