Despite a growing interest in epigenetics, performing proteomics studies of histone tail marks remains highly specialized. Mass spectrometry of histone tail marks is difficult due to the variety of modifications, coeluting isoforms, and dynamic range. Conventionally, these challenges have been met with database searching shotgun DDA, which detects histone marks but doesn’t provide accurate quantification; or optimizing high-level PRM methods, which accurately quantifies but cannot detect novel histone marks. Here, we have designed a robust histone DIA method and a flexible Skyline-based analysis workflow to more accurately and precisely quantify histone marks. Our targeted mass spectrometry Skyline-based workflow for quantifying histone tail modifications takes advantage of Skyline’s latest features, including staggered DIA isolation window demultiplexing to process raw data, the “Quantitative” fragment ion demarcation for site-localizing isobaric histone marks, and a retention time calculator that uses peptides co-enriched in the histone preparation as iRT anchors.

Orbitrap liquid chromatography-mass spectrometry. Peptides were analyzed by UPLC-MS (Thermo Dionex UPLC coupled with a Thermo Q-Exactive HF tandem mass spectrometer). We used an in-house pulled column created from 75 μm inner diameter fused silica capillary packed with 3 μm C18 beads (ReproSil-Pur, Dr. Maisch) to 30 cm. Solvent A was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 80% acetonitrile. For each injection, we loaded approximately 1 μg peptides and separated them using a 47-minute gradient from 2 to 35% B, followed by a 13 min washing gradient. For data dependent acquisition (DDA) analysis, a Top20 method was used (default charge state 2, minimum AGC target 5e4, charge exclusion 1 and >6, and dynamic exclusion 4s) with full MS (resolution 120,000; AGC 3e6, maximum IT 50 ms) and data dependent-MS2 (resolution 15,000; AGC 1e6, max IT 40 ms, isolation window 2.0 m/z, NCE 27)). For data independent acquisition (DIA) analysis, the mass spectrometer (Thermo Q-Exactive HF) was configured to acquire 25 × 24 m/z (covering 300-900 m/z) precursor isolation window DIA spectra (30,000 resolution, AGC target 1e6, maximum inject time 60 ms, 27 NCE) using an optimized staggered window pattern. Precursor spectra (target range ± 15 m/z at 60,000 resolution, AGC target 1e6, maximum inject time 60 ms) were interspersed every 51 MS/MS spectra. The isolation window scheme is detailed in Supplemental Table X on the PanoramaWeb project folder, XYZ DDA data analysis. Byonic (version 3.10.2) was used to search RAW files against a focused human or ant FASTA with histone proteins and contaminants (on the PanoramaWeb project folder, XYZ) within Proteome Discoverer (version 2.4) using the following parameters: arg-c digestion, 2 max missed cleavages, minimum peptide length 7; 8 maximum modifications; fixed carbamidomethyl on C and U, variable oxidation on M, variable acetylation on protein N-terminus and K, methylation on R, dimethylation on K and R, trimethylation on protein N-terminal A and K, phosphorylation on S, T, and Y, propionylation on peptide N-terminus and K, propionyl-methylation on K, and GG-propionylation on K; precursor mass tolerance 10 ppm, product mass tolerance 20 ppm; HCD fragmentation. DIA data analysis. Mass spectrometry data files were demultiplexed and converted to MZML using MSConvert (version 3.0.18). Database search results from DDA data analysis were built into a spectral library using Bibliospec [58] in Skyline-daily (version 20.1) [59]. DIA files were imported into Skyline using MS/MS IDs in the spectral library (all settings default except Peptide Settings: digestion enzyme ArgC [R|P], background proteome from Uniprot Dictyostelium FASTA (accessed 21 Dec 2020, 12742 entries); Transition Settings: Filter for precursor charges 1-3, ion charges 1-2, ion types y, b, p; from ion 1 to last ion; Full-Scan MS1 filtering with isotope peaks included (Count) and MS/MS filtering for DIA acquisition, centroid product mass analyzer, and Results only isolation scheme; use only scans within 5 minutes of MS/MS IDs).

The spectral library and iRT calculator were constructed using the stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry (Lin, Wein, Gonzales-Cope, Otte, Yuan, Afjehi-Sadat, Maile, Berger, Rush, Lill, Arnott, & Garcia, MCP 2014) .