Preparing Histones for Proteomics
Sydney Thomas based on protocols from Kim Krautkramer, last updated 9/2019
Step One: Extracting Histones
*All centrifugation after harvesting & washing should take place at 4°C.
*Add protease inhibitors to Buffer A before use. Stocks of protease inhibitors are in -20°C.
Cell/tissue lysis and crude fractionation (homogenization):
**Works well with ~4 X 106 cells or ~30 mg of tissue (have had success with as little as ~1-10 mg of fetal tissue!)**
- Pre-chill dounce homogenizer on ice before use
- If starting with cultured cells:
- Harvest 4 x 10^6 cells, wash twice in 1 mL ice-cold PBS.
- Pellet cells at 1000 x g, 1 min, discard supernatant.
- At this point you can also flash freeze cell pellets and store them at -80°C.
- If starting with frozen post-mortem tissue:
- For tough/fibrous tissues: (gut tissues, skeletal muscle) mince the frozen tissue using a razor blade in a small glass petri dish on ice with 400 uL buffer A. Cut the tip of a p1000 pipette tip and move the minced tissue to a pre-chilled 1mL glass dounce homogenizer (on ice). Use the remaining 400uL buffer A (800uL total) to wash the petri dish contents. Transfer wash to the douncer.
- For other tissues: (brain, kidney, heart, etc) (Note: Some tissues (white adipose and brain) have a ton of fat, and it’s hard to solubilize them without detergent. Solubilize by adding 0.2% NP-40 alternative to Buffer A) Cut the frozen tissues on dry ice (Note: Use the top of a tip box on dry ice, wash between sample groups. It’s important to keep tissues frozen during this step) into smaller chunks and dump the pieces directly into a pre-chilled dounce homogenizer (1mL, on ice) pre-filled with 800 uL buffer A.
- Transfer cell suspension/tissue to dounce homogenizer (Note: Try to make as few bubbles as possible during homogenization. Bubbles can break nuclei apart and reduce your yield. Store loose and tight pestles in a 50 mL tube on ice. Wash douncer and pestle with milliQ water between each sample. You can also use EthOH to wash, but never bleach or detergent). Perform enough strokes to effectively lyse the cells
- Recommendations: typically 20 with the loose pestle (just tissue) + 20 with a tight pestle is sufficient (both tissue and cells)
- If homogenizing cultured cells:
- Transfer homogenate to a new 1.5 ml epi tube if from cultured cells.
- If homogenizing tissue:
- Strain homogenate (Note: Strainers go in 50 mL tubes. You can use a pipette tip to pull the liquid through the strainer if you’re impatient. Also put out a beaker with diH2O to place strainers in once used. These strainers can eventually be washed with diH2O and dried) through a 100um nylon mesh strainer into either a 15mL or 50mL conical tube.
- Rinse the dounce homogenizer with 200 ul buffer A and add to tube containing the sample.
- Centrifuge at 800 x g for 10 minutes at 4°C.
- Supernatant = crude cytosolic fraction (cytosol + mitochondria)
- Pellet = crude nuclear fraction
- Wash nuclei pellet in 200-500 uL of ice-cold PBS and spin at 800 x g for 5-10 min (do this twice for tissue).
- If purifying histones from nuclei, you may immediately proceed to acid extraction or freeze the nuclei pellet and store at -80°C.
Step Two: Acid Extraction of Histones
You can start at this step directly with low volume samples!
- Slowly add 500 uL 0.4 N H2SO4 to the nuclei pellet; resuspend the pellet by gentle pipetting to break up the pellet.
- Incubate the sample with constant rotation or gentle shaking for 2-4h at 4°C.
- For sample beginning with more than 500 uL cell pellet, a 2h extraction is enough incubation time. Longer extraction is not recommended because basic proteins other than histones will also be extracted.
- For small sample size (<100 ul cell pellet), 4-h extraction will give a better yield.
- To remove nuclear debris:
- Centrifuge at 3400 x g for 10 min at 4°C.
- Transfer supernatant to a new 1.5- or 15-ml tube, based on volume.
- Pellet = nucleoplasm
- Supernatant = histones
4. Add 125 uL 100% TCA to supernatant and mix.
5. Let this mixture precipitate on ice for at least 1 hour to overnight.
- Do not disturb the precipitation.
6. Centrifuge at 3400 x g for 5 min at 4°C.
- Histones will form a “film-like” layer around the bottom of the tube, and the white pellet at the bottom contains mostly other proteins or nonprotein material.
- Carefully remove (and discard) the supernatant by aspiration without touching the precipitated proteins.
7. Rinse the tube with cold 100% acetone to cover the precipitated proteins.
8. Centrifuge at 3400 x g for 2 min at 4°C; discard supernatant.
- Instead of centrifuging, you can invert the tube and pour off supernatant.
9. Repeat steps 7-8.
10. Air-dry pellet for 30 min to overnight at RT until completely dry.
11. Dissolve the histones with ddH2O; make sure the “film-like” layer is completely dissolved. Typically, 100 uL ddH2O is used.
12. Centrifuge the tube containing reconstituted histones at 3400 x g or higher for 2 min.
13. Transfer supernatant to a new tube. Discard the pellet which contains mostly nonhistone proteins and other material.
14. Measure the protein concentration by Bradford (or other) method. Transfer 5 ug histone to new tube and dry down.
- The minimum amount of histone to perform mass spec is 1 ug
- Some people prefer to use 10 ug histone, if so, double the volumes in the subsequent steps.
15. Purified reconstituted or dried histones can be stored at -20°C.
Step Three: Trypsinization and Propionylation
- Add 10 uL 100 mM TEAB to 5 ug dried-down histones.
- You can also start with 10 ug histones. If so, increase all volumes x2.
- Check pH of solution. If needed, bring pH up to ~9-10 with ~1-2 uL 0.02M NaOH.
- Samples at high pH will label inefficiently
- Use 0.3 uL sample to spot onto pH paper (roughly equivalent to just dipping p10 tip into sample and touching onto pH paper)
- Mix propionic or heavy acetic anhydride 1:50 with ddH2O.
- Important: overlay anhydrides with nitrogen gas and wrap with parafilm after each use to prevent hydrolysis
- Add 1uL of anhydride:H2O mixture to the histone sample.
- Incubate at room temp for 2 min.
- Quench with 1uL 80 mM hydroxylamine. Incubate for 20 min at room temp.
- Check pH again. Trypsin is also inefficient at high pH.
- Trypsinize for 4 hours-overnight with 0.1 ug trypsin at 37°C.
- Trypsin is stored in 5 uL aliquots. To add 0.1 ug, add 5 uL water to 5 uL trypsin, then add 0.4 uL to each sample
- It’s best to keep the amount of time you incubate samples in trypsin similar for the sample sets you plan to compare.
Step Four: PIC-labeling and C18 Stage Tip
Note: Phenyl-isocyanate is extremely and acutely toxic. Absolutely DO NOT use this chemical without first carefully reading the MSDS, wearing proper PPE (including eye, skin, and respiratory protection) (Note: Protection for PIC includes two sets of gloves, lab coat, goggles, and a respirator. All tips go in PIC waste container), and having a waste plan in action. All tip/tube and liquid waste must be disposed of properly by chemical safety. Keep solid and liquid waste separate.
Never open PIC bottle outside of the hood!
FROM THIS POINT ONWARD ALL STEPS MUST BE PERFORMED IN THE FUME HOOD!
Move heat block and RT microcentrifuge into hood to perform these steps
- Prepare a 4% (v/v) phenyl-isocyanate in ACN (Note: At this point, all buffers should be made with mass-spec grade reagents).
- Add 3 uL of PIC/ACN to trypsinized histone peptides (Note: PIC is stored under the hood in a secondary container, and has parafilm around the cap. To put away, cover PIC with N2 and wrap in parafilm).
- Incubate at 37°C for 1 hour.
While you’re waiting:
Prepare stage-tips:
- Using large bore, non-beveled needle + syringe + spring thing, punch out enough discs for your sample amount (2-3 works well).
- Eject and press firmly (but not too hard) into a 200uL pipette tip. Ensure that the discs are securely wedged (Note: Don’t pack stage tips too tight, or they’ll take forever to get through) near the bottom of the p200 tip and that there is no space between discs.
At this point, you don’t need to use respirator, but still perform all steps in hood.
3. After 1 hour, acidify samples with 8 uL 1% TFA (Note: It’s common to see a white precipitant at this step).
4. Spin down histone samples at max speed for 3 minutes to pellet out any precipitate.
Stage Tip Desalting
- Conditioning – wet the disks by passing 30uL methanol through the stage tip.
- Pipette 30 uL onto the stage tip
- Centrifuge at room temp, 2000 x g, ~1 min
- Check to see if there is any liquid remaining above the disks, if so, spin a few more seconds (15 seconds a piece) until the last methanol has just left the tip. Do not let stage tips sit without buffer for excessive amounts of time – they need to be kept wet.
- Pre-wash disks – pass 30uL elution buffer through the tip as in step 1.
- Equilibrate disks – pass 30uL Equilibration/Wash buffer through the tip as in step 1.
- Really check whether everything passed through each tip at this step!
- Bind sample – pass sample through stage tip by pipetting the entire sample onto the tip and then centrifuging at room temp, 2000 x g for 1 min.
- Wash sample – pass 30uL Equilibration/Wash buffer through the tip as in step 1.
- Really check whether everything went through on this step as well.
- Transfer & elute – Transfer the stage tip to the new tube. At this point you don’t need to worry about PIC.
- pass 30uL Elution Buffer through the stage tip as in step 1
- When all sample has eluted, remove the tip and dry down samples completely using the SpeedVac. Make sure something eluted into each sample.
- Resuspend samples in 30 uL sample diluent. Spin down at max speed to remove white precipitant before transferring to UPLC tube.
Buffers
For Step one:
Buffer A*
10 mM Tris-HCl
10 mM NaCl
3mM MgCl2
pH 7.4
Buffer A Recipe (500 mL)
785 mg Tris-HCl
290 mg NaCl
142 mg MgCl2
*Add the following inhibitors buffer A:
histone deacetylase inhibitors: (aliquots stored at -20°C)
10mM nicotinamide (stock solution 2.5M = 250X)
1mM sodium-butyrate (stock solution 1M = 1000X)
4uM trichostatin a (TSA) (stock solution 4mM = 1000X)
Protease inhibitors:
10 ug/ml leupeptin (stock solution 10mg/mL = 1000X)
10 ug/ml aprotinin (stock solution 10 mg/mL = 1000X)
100 uM PMSF (stock solution 100mM = 1000X) (in DMSO, will not thaw on ice)
Note: For example, if you’re making 5 mL Buffer A, you will add 5 uL of everything but NAM. Add 20 uL of NAM to 5 mL Buffer A.
For Step Two:
PBS
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO42H2O
2 mM KH2PO4
Trichloroacetic acid (TCA)
[100% (w/v) in ddH2O]
Add 227 ml H2O to 500 g TCA
Add 10 mL H2O to 22.02 g TCA
H2SO4
0.4 N (0.2 M) in ddH2O
(Stock is 18.7 M, add 267 uL H2SO4 to 24.6 mL H2O)
For Step Three:
80mM NH2OH
50% wt stock solution = 16.32 molar solution
for 1mL solution: 4.9uL stock + 995uL ddH2O
For Step Four: (all LC-MS grade reagents!)
Conditioning Solution: 100% Methanol
Elution Buffer: 80% Acetonitrile (ACN), 0.5% TFA in H2O (16 mL ACN, 100 uL TFA, 4 mL H2O)
Wash/Equilibration Buffer: 0.5% TFA acid in H2O (100 uL TFA in 20 mL H2O)
Sample Diluent: 5% ACN, 0.1% acetic acid in H2O (1 mL ACN, 20 uL acetic acid, 19 mL H2O)