Decrypting lysine deacetylase inhibitor action and protein modifications by dose-resolved proteomics.

Lysine deacetylase inhibitors (KDACis) are approved drugs for cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and multiple myeloma, but many aspects of their cellular mechanism of action (MoA) and substantial toxicity are not well understood. To shed more light on how KDACis elicit cellular responses, we systematically measured dose-dependent changes in acetylation, phosphorylation, and protein expression in response to 21 clinical and pre-clinical KDACis. The resulting 862,000 dose-response curves revealed, for instance, limited cellular specificity of histone deacetylase (HDAC) 1, 2, 3, and 6 inhibitors; strong cross-talk between acetylation and phosphorylation pathways; localization of most drug-responsive acetylation sites to intrinsically disordered regions (IDRs); an underappreciated role of acetylation in protein structure; and a shift in EP300 protein abundance between the cytoplasm and the nucleus.

Prediction models combining zonulin, LPS, and LBP predict acute kidney injury and hepatorenal syndrome-acute kidney injury in cirrhotic patients.

The development of acute kidney injury (AKI) and hepatorenal syndrome-acute kidney injury (HRS-AKI) in cirrhosis has been associated with intestinal barrier dysfunction and gut-kidney crosstalk. We use the related markers such as zonulin, lipopolysaccharides (LPS), and lipopolysaccharide-binding protein (LBP) to predict AKI and HRS-AKI in cirrhotic patients and evaluate their in vitro effects on intestinal (Caco-2) cells and renal tubular (HK-2) cells. From 2013 to 2020, we enrolled 70 cirrhotic patients and developed prediction models for AKI and HRS-AKI over a six-month period.

Chemoproteomic target deconvolution reveals Histone Deacetylases as targets of (R)-lipoic acid.

Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic (R/S)-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, (R/S)-lipoic acid is an approved drug for the treatment of diabetic neuropathy.

Preservation of vascular endothelial glycocalyx and barrier by activation of adenosine A2A receptor (AR) improved renal dysfunction in cirrhotic rats.

Cirrhosis-related hepatic and renal endothelial dysfunction is characterized by macrophage-endothelium adhesion-mediated inflammation, glycocalyx/barrier damage, and impaired vasodilation. Activation of adenosine A2A receptor (AR) protects cirrhotic rats from impairment of hepatic microcirculation post hepatectomy. This study evaluates the effects of AR activation on the cirrhosis-related hepatic and renal endothelial dysfunction in biliary cirrhotic rats receiving two weeks of AR agonist PSB0777 [bile duct ligated (BDL)+PSB0777] treatment.

UHMK1 is a novel splicing regulatory kinase.

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated.

Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo.

Linking post-translational modifications and protein turnover by site-resolved protein turnover profiling.

Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs.

Evaluation of Disposable Trap Column nanoLC-FAIMS-MS/MS for the Proteomic Analysis of FFPE Tissue.

Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS).

A data-independent acquisition-based global phosphoproteomics system enables deep profiling.

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.

Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting.

Microflow liquid chromatography tandem mass spectrometry (μLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d.

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery.

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS.

Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.