Protocol for Decoding the Protein Composition of Whole Nucleosomes with Nuc-MS: Sample Preparation, Data Acquisition and Analysis

The disassembly and digestion of nucleosome particles in current proteomics approaches forfeits correlations among histones and blurs nucleosome-level information. We developed Nuc-MS which analyzes whole nucleosomes and displays their histone modi�cations and variants in a single mass spectrum. In this protocol, we provide step-by-step instructions for preparation of mononucleosomes for mass spectrometry (MS) analysis, and parameter sets for Nuc-MS data acquisition and data analysis.

2. Lyse cells by adding equal volume of Buffer A supplemented with 2% Triton X-100 a. Note: Final concentration of Triton X-100 in cell suspension is 1%.
3. Incubate on ice for 10 mins with occasional mixing.11.Supplement nuclei suspension with 2 µL of NEB micrococcal nuclease per 1 mL of cell suspension.
12. Incubate at 37C for 15 minutes with occasional mixing.13.To quench micrococcal nuclease digestion, incubate on ice and supplement nuclei suspension with 2 mM EGTA and 1 mM EDTA.
15. Incubate 15 minutes on ice with occasional mixing.
16. Centrifuge at 20,000 xg for 20 mins at 4C and collect resulting supernatant containing nucleosomes.17.Mononucleosomes are concentrated and buffer exchanged into Buffer A supplemented with 650 mM NaCl using 30 kDa MWCO spin lter (Millipore-Sigma).Sample Desalting for Nuc-MS 1. Puri ed mononucleosomes and synthetic nucleosomes (Nucs; e.g.EpiCypher designer Nucs) must be concentrated and desalted into 150 mM ammonium acetate solution.Best results are achieved using the 0.5 mL 30 kDa MWCO spin lter (Millipore-Sigma).A concentration >2 µM in a volume of 50 µL yields adequate signal intensity for MS 1-3 .

2.
Filter is rst conditioned by spinning 500 µL of 150 mM ammonium acetate for 3 mins at 13,000 xg.Upon completion of spin, empty the lter of any remaining liquid.
3. Add up to 500 µL of Nuc sample into the lter -topping off with ammonium acetate -and spin for 5 mins at 13,000 xg or until sample is concentrated at or below 100 µL. 4. For sample desalting, add ammonium acetate up to the 500 µL mark and spin sample for 5 mins at 13,000 xg.Repeat desalting process 10-15 times for best results.

Native electrospray ionization (nESI) can be achieved with commercial Nanospray and Nanospray Flex
Ion Sources with a static NSI probe (Thermo Fisher Scienti c) as well as a capillary-based ion source as described previously [1].4. Once stable electrospray is achieved, the MS 1 (intact mass of Nuc particle) can be collected using the following parameters in positive ion mode (QE-EMR ; QE-UHMR): h.*Note that histone ejection at the source using SID on the QE-EMR is not as e cient as IST on the QE-UHMR.A 50-150 V SID range is provided given that some DNA fragmentation occurs which must be balanced with histone ejection.generating a p-score.mMass generates in silico a list of theoretical fragment ions for a target proteoform and is thus used to interrogate individual fragment ions within a spectrum not identi ed by TDValidator or ProSight Lite.

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7 .
Pellet nuclei by centrifuging at 1,300 xg for 12 minutes at 4C to separate nuclei from cell debris.8.Resuspend nuclei pellet with 2X PCV of Buffer A. 9. Incubate at room temperature for 5 mins.10.Supplement nuclei suspension with 1 mM CaCl2.
18. Concentrated nucleosomes are puri ed using HiPrep™ 26/60 Sephacryl S-300 HR column (Size Exclusion Chromatography) equilibrated with Buffer A supplemented with 650 mM NaCl using AKTA Prime Plus FPLC (GE Lifescience).19.Fractions containing mononucleosomes are validated by purifying 10 ug of material using Qiagen DNA clean up kit and resolving it on 2% agarose gel in 0.5X TBE.a. Note: Fractions showing ~150 DNA bp are pooled together for Nuc-MS analysis.

5 .
For nal spin, centrifuge for 10 minutes to concentrate sample >2 µM in <50 µL.Nuc-MS Data Acquisition 1. Instrumentation: Given the high mass of Nuc particles, the Q Exactive HF mass spectrometer with Extended Mass Range (QE-EMR) and Q Exactive HF Ultra-High Mass Range (QE-UHMR) instrumentation are best for Nuc-MS analysis.Other instruments with up to 8000 m/z transmission and detection range may also be suited for Nuc-MS.2. XCalibur QualBrowser 4.0.27.10 (Thermo Fisher Scienti c) is used for MS data acquisition.