Site-Specific Lysine Acetylation Stoichiometry Across Subcellular Compartments.

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Simultaneous determination of trace concentrations of aldehydes and carboxylic acids in particulate matter.

Carboxylic acids and aldehydes are present in ambient air particulate matter (PM) originating from both primary emission and secondary production in air and may, due to their polarity have, an impact on formation of cloud condensation nuclei. Their simultaneous determination may provide improved understanding of atmospheric processes. We developed a new analytical method allowing for a single step determination of majority of carboxylic acids and aldehydes (+95 compounds). This sample preparation employed O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA·HCl) in methanol to yield oximes (for aldehydes) and methyl esters (for majority of acids); with the limits of detection of 0.02-1 ng per injection, corresponding to approximately 0.4-20 µg/gPM. Subsequent trimethylsilylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was employed only for aromatic acids, which were not completely esterified, and for hydroxyl groups. Our method, in contrast to previous primarily qualitative studies, based on derivatization with an aqueous PFBHA followed by BSTFA derivatization, is less labor-intesive and reduces sample losses caused by an evaporation. The method was tested with a broad range of functionalized compounds (95), including monocarboxylic, dicarboxylic and aromatic acids, ketoacids, hydroxyacids and aldehydes. The developed protocol was applied to wood smoke (WS) and urban air standard reference material 1648b (UA) PM. The observed concentrations of aldehydes were 10-3000 µg/gPM in WS PM and 10-900 µg/gPM in UA PM, while those of acids were 20-1800 µg/gPM in WS PM and 15-1200 µg/gPM in UA PM. The most prominent aldehydes were syringaldehyde and vanillin in WS PM and glyoxal in UA PM. The most abundant acids in both PM samples were short-chain dicarboxylic acids (≤C10). WS PM had a high abundance of hydroxyacids (vanillic and malic acids) as well as ketoacids (glutaric and oxalacetic) while UA PM also featured a high abundance of long-chain monocarboxylic acids (≥C16).

Identifying Dysregulated Epigenetic Enzyme Activity in Castrate-Resistant Prostate Cancer Development.

There is a tremendous need for novel strategies aimed at directly assessing activities of histone modifiers to probe epigenetic determinants associated with disease progression. Here, we developed a high-throughput peptide microarray assay to identify altered histone lysine (de)acetylation activity in prostate cancer (PCa). This microarray-based activity assay revealed up-regulated histone acetyltransferase (HAT) activity against specific histone H3 sites in a castrate-resistant (CR) PCa cell line compared to its hormone-sensitive (HS) isogenic counterpart. NAD+-dependent deacetylation assays revealed down-regulated sirtuin activity in validated CR lines. Levels of acetyltransferases GCN5, PCAF, CBP, and p300 were unchanged between matched HS and CR cell lines. However, autoacetylation of p300 at K1499, a modification known to enhance HAT activity and a target of deacetylation by SIRT2, was highly elevated in CR cells, while SIRT2 protein level was reduced in CR cells. Interrogation of HS and matched CR xenograft lines reveals that H3K18 hyperacetylation, increased p300 activity, and decreased SIRT2 expression are associated with progression to CR in 8/12 (66%). Tissue microarray analysis revealed that hyperacetylation of H3K18 is a feature of CRPC. Inhibition of p300 results in lower H3K18ac levels and increased expression of androgen receptors. Thus, a novel histone array identifies altered enzyme activities during the progression to CRPC and may be utilized in a personalized medicine approach. Reduced SIRT2 expression and increased p300 activity lead to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC.