TUM Kuster Lab - 2023_Chang_decryptM_KDACi

Decrypting lysine deacetylase inhibitor action and protein modifications by dose-resolved proteomics
Data License: CC BY 4.0 | ProteomeXchange: PXD047419 | doi: https://doi.org/10.6069/81v5-0w13
  • Organism: Homo sapiens
  • Instrument: Orbitrap Eclipse
  • SpikeIn: Yes
  • Keywords: Chemical proteomics, acetylation, phosphorylation, lysine deacetylase inhibitors, HDACs, proteomic pharmacology
  • Lab head: Bernhard Kuster Submitter: Miriam Abele
Lysine deacetylase inhibitors (KDACis) are approved drugs for cutaneous T-cell lymphoma (CTCL) and multiple myeloma. The drugs lead to increasing acetylation levels of histones and other proteins, altered gene expression, and cell death. To characterize the mechanisms of action of these drugs in more detail, we systematically measured dose-dependent drug-induced changes in protein expression, acetylation, and phosphorylation in response to 21 clinical and pre-clinical KDACis. The resulting 872,000 dose-response curves revealed, for instance, poor selectivity of HDAC1, 2, 3, and 6 inhibitors in cells, 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. This comprehensive dataset serves as a resource for the investigation of the molecular mechanisms underlying KDACi action in cells and can be explored online in ProteomicsDB.
Experiment Description
Selection and validation of target peptides: Based on the result of the DDA acquisition, we chose 27 precursors from 27 different peptides + 3 Procal peptides (for quality assessment) for PRM assay. LC-MS/MS measurement: Targeted measurements using Parallel Reaction Monitoring (PRM) were performed with a 50-min linear gradient (4% to 32% acetonitrile) on a Dionex Ultimate 3000 RSLCnano system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in PRM and positive ionization mode. MS1 spectra (280–1300 m/z) were recorded at a resolution of 60,000 using an AGC target value of 8×105 and a MaxIT of 50 ms. The precursor isolation window was set to 1.2 Th. Precursors were fragmented with HCD at 30% NCE. Targeted MS2 spectra were acquired at a resolution of 50,000, an AGC target value of 1×105, and a MaxIT of 90 ms. Data analysis with Skyline: We built an experimental library with all PRM raw files searched with MaxQuant and default parameters against a fasta file containing 31 synthetic peptides and 41 Procal peptides. PRM data were analyzed with Skyline (64-bit; version For all target peptides, the six most intense fragment ions were selected based on the experimental spectral library (built-in option). Raw PRM data was imported into Skyline and peak integration and transition interferences were reviewed manually. If necessary, integration boundaries were adjusted and interfered transitions were removed from the complete dataset. A minimum of four transitions per peptide were kept. MS-system carry-over was minimized throughout all sample measurements by performing measurements from shortest incubation times to longest incubation times. The intensities of deacetylated peptides were computed by summing up all (4-6) fragment ion intensities.
Sample Description
Based on dose-dependent acetylomic profiling, we synthesized 31 target acetylated peptides for validation, containing ac-K sites positioned at the center and flanked by seven amino acids on each side (JPT Peptide Technologies GmbH, Germany). The peptide pool was incubated either with recombinant HDAC1 or HDAC6 for 20, 60, 120, and 180 minutes. Samples were desalted prior to LC-MS/MS measurements. Further details can be found in the method section of the corresponding manuscript.
Created on 11/30/23, 2:28 PM
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Result_analysis_2023-11-28_11-06-40.sky.zip2023-11-30 14:28:3225252522116