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

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

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

Global Discovery and Temporal Changes of Human Albumin Modifications by Pan-Protein Adductomics: Initial Application to Air Pollution Exposure

Assessing personal exposure to environmental toxicants is a critical challenge for predicting disease risk. Previously, using human serum albumin (HSA)-based biomonitoring, we reported dosimetric relationships between adducts at HSA Cys34 and ambient air pollutant levels (Smith et al., Chem. Res. Toxicol. 2021, 34, 1183). These results provided the foundation to explore modifications at other sites in HSA to reveal novel adducts of complex exposures. Thus, the Pan-Protein Adductomics (PPA) technology reported here is the next step toward an unbiased, comprehensive characterization of the HSA adductome. The PPA workflow requires <2 μL serum/plasma and uses nanoflow-liquid chromatography, gas-phase fractionation, and overlapping-window data-independent acquisition high-resolution tandem mass spectrometry. PPA analysis of albumin from nonsmoking women exposed to high levels of air pollution uncovered 68 unique location-specific modifications (LSMs) across 21 HSA residues. While nearly half were located at Cys34 (33 LSMs), 35 were detected on other residues, including Lys, His, Tyr, Ser, Met, and Arg. HSA adduct relative abundances spanned a ∼400 000-fold range and included putative products of exogenous (SO2, benzene, phycoerythrobilin) and endogenous (oxidation, lipid peroxidation, glycation, carbamylation) origin, as well as 24 modifications without annotations. PPA quantification revealed statistically significant changes in LSM levels across the 84 days of monitoring (∼3 HSA lifetimes) in the following putative adducts: Cys34 trioxidation, β-methylthiolation, benzaldehyde, and benzene diol epoxide; Met329 oxidation; Arg145 dioxidation; and unannotated Cys34 and His146 adducts. Notably, the PPA workflow can be extended to any protein. Pan-Protein Adductomics is a novel and powerful strategy for untargeted global exploration of protein modifications

Additional file 1 of De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a

Additional file 1: Figure S1. EHMT1/GLP and EHMT2/G9a de novo methylate H3K18, H3K23, and H3K27, in vitro. (A) Annotated spectra, ions counts and mass error tables for the mass spectrometry analysis of EHMT1/GLP and EHMT2/G9a in vitro HMT reactions (B) Western blot panel of EHMT1/GLP and EHMT2/G9a in vitro HMT reactions for various H3K18 and H3K27 methylation states. H3K4me3 and H3K36me3 were included as negative controls. 50B11 whole cell lysate was used to validate that antibodies for H3K27me1/2, H3K4me3, and H3K36me3, were functional given these modifications were not detected by western blotting in EHMT1/GLP and EHMT2/G9a in vitro HMT reactions. Figure S2. Pharmacologic inhibition of EHMT1/GLP and EHMT2/G9a decreases H3K18 and H3K27 methylation in vitro. (A) UNC0642 dose titration with recombinant SetDB1 and Suv39h2, as measured by the MTase-Glo assay. (B) Western blotting panel showing production and inhibition of various H3K18 and H3K27 methylation states by EHMT1/GLP and EHMT2/G9a. Unlike H3K27me3, H3K27me1 and H3K27me2 were undetectable in this in vitro assay. Figure S3. Pharmacologic inhibition of EHMT1/GLP and EHMT2/G9a decrease H3K18 and H3K27 methylation in vivo. (A) Western blot panel of H3K9, H3K18, H3K23 and H3K27 methylation states in MC38 cells treated with DMSO or UNC0642. (B) Western blot panel of H3K18 and H3K27 methylation states of 50B11 cells treated with DMSO or UNC0642 (C)Western blot panel of H3K18 and H3K27 methylation states of HEK293 cells treated with DMSO or UNC0642. Figure S4. H3K9M, but not H3K23M, decrease H3K23me3 in vivo. (A) MC38 and (B) 50B11 cells lines were transduced with lentivirus carrying plasmids encoding various lysine (K) to methionine (M) mutations at specific residues (e.g. (, 23, 27, etc.) to evaluate their corresponding effect on various histone H3 methylation state in vivo. Figure S5. Genetic ablation of GLP, G9a or both perturbs H3K18 and H3K27 methylation in vivo. Western blot of H3K18 and H3K27 methylation states in mouse ESCs knocking down either EHMT1/GLP, EHMT2/G9a or both and their corresponding effect on H3K18 and h3K27 methylation states. Figure S6. Antibody validation via ELISA and peptide competition assays. Using a 96-well plate ELISA format, ELISAs and peptide competition assays were done to validate H3K9me1, H39me2, H3K9me3, H3K18me1, H3K18me2, H3K18me3, H3K23me1, H3K23me2 H3K23me3, H3K27me1, H3K27me2, and H3M27me3 antibodies for their target methylation state, cross-reactivity with other methylation states of the same epitope and cross-reactivity with methyl-lysines of a different epitope (e.g. K9 antibodies with K23-methyl peptides, etc.)

Post-Translational Modifications (PTMs), Identified on Endogenous Huntingtin, Cluster within Proteolytic Domains between HEAT Repeats

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington’s disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD

Proteomic Analysis of Chinese Hamster Ovary Cells

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682 097 MS/MS spectra, 93 548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included <i>N</i>-acetylation modifications, and 1292 different proteins were observed to be <i>N</i>-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions