Protocol 1: Histone extraction (HLB)
1. Resuspend the cells in cold Phosphate Buffered Saline (PBS): 1*106 cells/50µl.
2. Distribute in 1.5 mL Eppendorfs: 200µl = 4*106 cells
3. Spin down for 10minutes at 4°C and 300g
4. Remove the supernatant
5. Add 800 µL Hypotonic Lysis Buffer (HLB) to the cell pellet (200 µL for 1*106 cells)
6. Prepare HLB buffer: 10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2 supplemented with 1 mM DTT, Halt Protease and Phosphatase Inhibitor Cocktail 100x (78440) and phosphatase inhibitor cocktails II and III (P5726 and P0044, Sigma-Aldrich, 1 mL of cocktail for 100 mL of buffer) (See Table 1 for quantities).
7. Rotate for 30minutes at 4°C to promote lysis of cell membrane (mechanical shear)
8. Pellet the nuclei in centrifuge for 10minutes at 4°C and 10 000 g
9. Discard supernatant
10. Resuspend the pellet in 125 µL (for 1*106 cells) 0.4N HCl by soft pipetting until no clumps left in solution (if necessary: vortex)
11. Incubate 30minutes in acid on rotator at 4°C to promote lysis of nuclei and solubilization of histones
12. Spin down for 10minutes at 4°C and 16000 g.
13. Transfer supernatant to new Eppendorf (histones are present in the acid since they are alkaline proteins)
14. Add, drop by drop, TCA until a final concentration of 33% is reached to promote precipitation of histones and invert the tube several times (results in a milky solution)
15. Incubate on ice for 30minutes
16. Spin for 10minutes at 4°C and 16000 g to pellet the histones
17. Remove the supernatant (be careful: the pellet is not always visible)
18. Add ice-cold acetone (do not resuspend the pellet) to remove TCA, make sure the pellet is fully covered with acetone
19. Spin for 5minutes at 4°C and 16000 g
20. Remove the supernatant
21. Add cold acetone again (do not resuspend the pellet) to remove TCA.
22. Spin for 5minutes at 4°C and 16000 g
23. Remove the supernatant
24. Dry at room temperature for 30minutes (until no acetone left)
25. Resuspend in MilliQ water (50 µl for 1x106 cells)
26. Transfer 400.000 cells (20 µl) to a new Eppendorf tube for gel-electrophoresis (optionally) (If there are still clumps left: Spin for 10minutes at 4°C and 16000 g and transfer the supernatant in a fresh Eppendorf)
27. Vacuum dry the samples (centrivap)
Protocol 2: Gel-electrophoresis
Sample preparation
1. Dry sample (equal to 400.000 cells)
2. Resuspend samples in 10µl laemmli-buffer
3. Add 1µl β-mercaptoethanol in a fume hood to each sample
4. Vortex and spin down
5. Incubate for 7minutes at 95°C in a thermoshaker
6. Spin down
Prepare Criterion Cell
7. Place the criterion cell on ice in a fume hood
8. Remove the sticker from the bottom of the gel cassette and check the gel for cracks
9. Put the gel cassette in the criterion cell
10. Fill the reservoir with running buffer (25mM Tris, 0.1% SDS, and 192mM glycine in MilliQ water) and take out the comb
Running of the samples
11. Load the samples and standards (2 µg of bovine histones) on the gel (3 standards per gel: lane 1, lane 9 and lane 18)
12. Put the cover on the criterion cell
13. Start running the gel on 200V
14. Stop running when the frontline is almost gone
Visualization
15. Take out the cassette
16. Incubate in fixation-solution (7% acetic acid, 10% methanol in MilliQ water) for 10minutes on a shaker
17. Wash the gel 3 times for 5minutes in MilliQ water on a shaker
18. Incubate in SyproRuby overnight
19. Wash the gel 3x for 10minutes in MilliQ water on a shaker
20. Visualize the gel by using a Versadoc
Protocol 3: Propionylation and tryptic digestion
Propionylation before
1. Vacuum dry the samples (20 µg/sample)
2. Add 20 µL TEAB (1M)
3. Add 20 µL Prop-reagent (Isopropylalcohol:propionic anhydride (79:1))
4. Spin down & Incubate at room temperature for 30minutes
5. Add 20 µL H2O
6. Spin down & Incubate at 37°C for 30minutes
7. Vacuum dry samples
Trypsin digest (Final Volume: 50µl)
8. Add 500 mM TEAB
9. Add CaCl2 and ACN
a. Final conc CaCl2: 1mM
b. Final conc ACN: 5%
10. Resuspend trypsin in 500 mM TEAB
11. Add trypsin at a 1:20 ratio (w/w) => 1 µg trypsin/20 µg histones
12. Spin down & incubate overnight at 37°C
13. Vacuum dry samples
Propionylation after
14. Vacuum dried samples (20 µg/sample)
15. Add 20 µL TEAB (1M)
16. Add 20 µL Prop-reagent (Isopropylalcohol:propionic anhydride (79:1))
17. Spin down & Incubate at room temperature for 30minutes
18. Add 20 µL H2O
19. Spin down & Incubate at 37°C for 30minutes
20. Vacuum dry samples
Reversing overpropionylation hydroxylamine mediated
21. Vacuum dried samples (20 µg/sample)
22. Add 50 µL 0.5 M NH2OH
23. Add 15 µL NH4OH at pH 12
24. Spin down & Incubate at room temperature for 20minutes
25. Adjust pH with formic acid: 30 µl 100% FA
26. Vacuum dry samples
Data-dependent acquisition mass-spectrometry
1. The propionylated samples, complemented with a Beta-Galactosidase (ß-gal) (Sciex) are resuspended in 0,1% FA resulting in 1.5 μg histones and 50 fmol ß-gal in a 9 µL injection.
2. A quality control (QC) mixture is created by combining 2 μL of each sample.
3. The samples are acquired on a mass spectrometer according to the parameters in Parameter file 1.
Data-analysis of hPTMs
1. For all runs, raw data is imported in Progenesis QIP 4.2. (Nonlinear Dynamics, Waters) followed by alignment, feature detection, and normalization.
2. Next, an ∗.mgf file is created based on the twenty MS/MS spectra closest to the elution apex and exported for searches using Mascot (Matrix Science).
3. First, a standard search is performed on the exported *.mgf file to identify non-propionylated standards (ß-gal and MPDS) and to verify underpropionylation (Parameter file 2).
4. Second, to identify the proteins present in the sample and to detect unexpected histone post-translational modifications, an error-tolerant search with fixed K- and N-term propionylation without biological modifications is carried out against a complete Human Swissprot database (downloaded from Uniprot and supplemented with contaminants from the cRAP database (https: //www.thegpm.org/crap/)) (Parameter file 3).
5. Subsequently, a FASTA database is created based on the results of the error-tolerant search.
6. Next, the three MS/MS spectra closest to the elution apex per feature are merged into a single ∗.mgf file and exported for a Mascot-search including the following parameters: 1) a mass error tolerance of 10 ppm and 50 ppm for the precursor ions and the fragment ions respectively; 2) Arg-C enzyme specificity, allowing for up to one missed cleavage site; 3) variable modifications included acetylation, butyrylation, crotonylation, and trimethylation on K, methylation on R, dimethylation on both K and R, deamidation on asparagine (N), glutamine (Q) and R (the latter representing citrullination), phosphorylation on serine (S) and threonine (T), and oxidation of methionine (M); and 4) fixed modifications included N-terminal propionylation and propionylation on K (Parameter file 4).
7. The search is performed against the above-mentioned custom-made FASTA database.
8. This Mascot result file (∗.xml-format) is imported into Progenesis QIP 4.2 for annotation.
9. To resolve isobaric near-coelution, features that are identified as histone-peptidoforms are manually validated and curated.
10. To correct for variations in sample loading, samples are normalized against all histone peptides.
11. Outlier detection and removal are done based on the principal component analysis (PCA).
12. Finally, the deconvoluted peptide ion data of all histones can be exported from Progenesis QIP 4.2 for further analysis.
A fully detailed description of the use of Progenesis QIP for histone analysis can be found in Provez et al. (https://www.biorxiv.org/content/10.1101/2022.05.05.490796v1)
Acid extractome analysis after histone extraction
Next to histones, other alkaline proteins remain in the HCl during acid extraction. For this purpose, we export the protein data from Progenesis QIP and use the MSqRob software for statistical analysis. Herein, relative protein quantification is done by implementing the peptide-level robust ridge regression method.
1. First, the deconvoluted peptide ion data of all identified peptides is exported from Progenesis QIP 4.2.
2. MSqRob requires an annotation file that contains the name of the runs included in the experiment and as well as the condition to which each run belongs (i.e. naïve or primed).
3. Log transformation and quantile normalization of the data are performed by the software.
4. Finally, pairwise comparisons (e.g. naive versus primed) are carried out and the result files can be exported for further use.