Max Perutz Labs MS Facility - Seiser - Histone acetylation in HAP cells

Relative quantification of acetylated histone peptides in HAP1 cells after treatment with MS-275
Data License: CC BY 4.0 | ProteomeXchange: PXD030272 | doi:
  • Organism: Homo sapiens
  • Instrument: Q Exactive HF-X
  • SpikeIn: No
  • Keywords: Chromatin, histone deacetylase, acetylome, HDAC inhibitor
  • Lab head: Christian Seiser Submitter: Markus Hartl
In a previous exploratory mass spectrometry experiment, we unravelled multiple histone and non-histone deacetylation targets of class I HDACs following acetyl-enrichment. Here, using targeted mass spectrometry without prior acetyl-enrichment, we validated selected histone targets in HAP1 cells treated for various time points with the HDAC1, HDAC2 and HDAC3 specific inhibitor MS-275. We confirmed significantly increased signals upon MS-275 treatment for 20 out of 21 selected acetylated peptides of histones H2A.V, H2B type 1-K/1-F, H3.3 and H4. The abundance of most of the selected acetylated peptides was increased already upon 6 hours of MS-275 treatment, and further increased after 24 hours. In summary, this validation experiment demonstrates the reliability of previous targeted approach and gives an additional insight in the the dynamics of acetylation increases following catalytic inactivation of HDAC1, HDAC2 and HDAC3.
Experiment Description
Based on data from a previous experiment, we selected peptides that showed increased lysine acetylation upon HDAC inactivation for validation using label-free targeted MS. For normalization to total protein levels, additional peptides that were not found to be modified (experimentally or according to UniProt) were additionally selected. The selection of the peptides was performed with Skyline (MacLean et al., 2010). Samples for the validation experiment included untreated control cells and cells treated for various time points with 3 µM MS-275 (2, 6 and 24 hours). Following sample preparation, peptide samples were subjected to nano-LC-MS/MS, applying a segmented linear gradient from 2% to 35% and finally 80% solvent B (80 % acetonitrile, 0.1 08 % formic acid; solvent A, 0.1 % formic acid) at a flow rate of 230 nLnl/min over 60 min. Parallel-reaction monitoring (PRM) data acquisition was performed using a scheduled method with 2-5 min windows for each target based on the retention time determined from a prior DDA LC-MS/MS run of a test sample (conditions: HAP1, 3 µM MS-275, 24 hours). Raw data were obtained using an Orbitrap Q-Exactive HF-X (Thermo Fisher Scientific) mass spectrometer applying the following settings: survey scan with MS1 45 k resolution, AGC 3E6, 50 ms IT, over a range of 340 to 1000 m/z, PRM scan with 60 k resolution, AGC 2E5, 400 ms IT, isolation window of 0.7 m/z, and NCE of 28%. MS1 signals of iRT standards (Biognosys) were used as quality control for monitoring instrument performance in Skyline. Data analysis, manual validation of all transitions (based on retention time, relative ion intensities, and mass accuracy), and relative quantification was performed in Skyline. Three specific transitions were selected for each peptide and their peak areas were summed up for peptide quantification (total peak area). Unmodified peptides were used for calculating a normalization factor for each sample, which was finally used to normalize the intensities of modified peptides.
Sample Description
2E+07 cells were prepared in lysis buffer containing 8 M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF, 5 mM sodium butyrate, 10 ng/ml NAM, 10 ng/ml trichostatin A (TSA) and 1x cOmplete protease inhibitor cocktail (+EDTA). 250 U of benzonase (Merck) were added to each sample. Lysis was achieved using a sonication device (Bioruptor, Diagenode). Following lysis, cells were centrifuged at 15,000 rcf for 10 min at 4 °C to remove insoluble material. The protein fraction was precipitated by addition of 4x volumes of cold (- 20°C) 100% acetone and 4°C incubation overnight. The protein pellet was washed with cold (-20°C) 80% acetone, air dried for 5 min, and resolved in 8 M urea, 50 mM ammonium bicarbonate buffer (ABC). Protein concentration was determined using the Pierce 660 nm Protein Assay (Thermo Scientific). Proteins were reduced with 10 mM DTT for 45 min at RT, and subsequently carbamidomethylated with 20 mM iodoacetamide for 30 min at RT in the dark. The alkylation reaction was quenched by adding 5 mM DTT for 10 min. Prior to digestion, the urea concentration was reduced to 4 M with 50 mM ABC. 50 µg protein per samples were incubated for 90 min at 37 °C with Lys-C (Wako Laboratories) at an enzyme-to-substrate ratio of 1:100 and further digested overnight at 37 °C with sequencing grade trypsin (Promega) at an enzyme-to-substrate ratio of 1:50. Following digestion, samples were acidified using 10% TFA (trifluoroacetic acid) (Sigma-Aldrich) to a final concentration of 0.5%, and desalted on a 50 mg tC18 Sep-Pak cartridge (Waters). Desalted samples were dried for 30 min in a SpeedVac concentrator and subsequently lyophilized overnight.
Created on 12/8/21, 8:56 PM
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