TITLE: Tissue Homogenization and GELFrEE for Top-Down Proteomics
AUTHORS: Bryon  Drown, Jeannie  Camarillo, Cameron Lloyd-Jones, Neil  Kelleher

[DESCRIPTION]
Solid human tissue is homogenized, protein is extracted, and samples prefractionated and desalted. Protein samples are ready for top-down proteomics.

[GUIDELINES]
Text written in italic involves critical steps, important observations, relevant information and safety notes. 


[STEPS]
SECTION: Cell lysis, protein precipitation, and lysate quantitation
1. Cryopulverize tissue sample with Retsch Cryomill
 

SECTION: Protein fractionation by GELFrEE
14. Transfer the calculated volume containing 300 µg of protein to a new 1.5-mL Protein LoBind tube. The leftover lysates in 1% (wt/vol) SDS can be stored for several months at −80 °C. From this point on, keep the sample tubes on ice until immediately before GELFrEE. 
SECTION: Protein fractionation by GELFrEE
13. Calculate 300 ug protein
SECTION: Protein fractionation by GELFrEE
15. Add 8 µL of 1 M DTT, 30 µL of GELFrEE sample loading buffer, and dH2O to a final volume of 150 µL.
SECTION: Protein fractionation by GELFrEE
16. Vortex each tube for 10–20 s, and heat it at 95 °C for 5 min. Centrifuge the samples at 1,000g for 2 min at 4 °C to re-collect any condensation at the tube lids after heating
SECTION: Protein fractionation by GELFrEE
17. Perform fractionation by GELFrEE by following the manufacturer’s protocol for 10% cartridges. 
 

SECTION: Protein fractionation by GELFrEE
18. After fractions 1-5 of each sample has been collected into a new, 1.5-mL Protein LoBind tube, the fracationation process can end or more fractions can be collected, depending on the experiment.This is a natural stopping point in the protocol, as GELFrEE fractions are stable for several months at −80 °C.
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
19. Mix 5–10 µL of each fraction of interest with the appropriate amount of Laemmli sample buffer for the gel lane capacity in a new microcentrifuge tube. Boil these gel samples at 95 °C for 5 min and spin down (5,000g, 20–25 °C, 30–60 s). 
SECTION: Cell lysis, protein precipitation, and lysate quantitation
2. Transfer pulverized tissue to a 15 mL conical tube and resuspend in 2 mL of cold RIPA cell lysis buffer (including 1x HALT protease/phosphatase inhibitor cocktail).
SECTION: Cell lysis, protein precipitation, and lysate quantitation
5. Transfer two 200 uL aliquots from each sample lysate to fresh 1.5mL Lo-bind Eppendorf tubes


SECTION: Cell lysis, protein precipitation, and lysate quantitation
6. Add 6 volumes of ice-cold acetone to the lysates, vortex at high speed (setting 8) for 10–20 s, and place the tubes in a −80 °C freezer for at least 30 min to precipitate the proteins. The protein precipitate is stable in acetone overnight. 
 

SECTION: Cell lysis, protein precipitation, and lysate quantitation
7. Remove the tubes from the freezer and immediately centrifuge 21000 x g, 30 min, 4 °C  

SECTION: Cell lysis, protein precipitation, and lysate quantitation
8. Remove as much acetone as possible from the protein pellets without disturbing them. Place tubes in a biological safety cabinet for 5–10 min to evaporate off any excess acetone.
SECTION: Cell lysis, protein precipitation, and lysate quantitation
9. Resuspend the protein pellet in 500 µL of 1% (wt/vol) SDS solution with pipetting and probe sonication It may take multiple rounds of vigorous pipetting to completely dissolve the precipitates. Lysates my also be stored at -80C until further processing.
SECTION: Cell lysis, protein precipitation, and lysate quantitation
10. Transfer 25 µL of the solution to a new 1.5-mL Protein LoBind tube for the BCA assay. The tubes containing the remaining 200 µL of resuspended proteins should be kept on ice.
SECTION: Cell lysis, protein precipitation, and lysate quantitation
11. A BCA assay should now be performed on the 25-µL aliquots of sample proteome, using a 96-well plate and following the manufacturer’s recommended protocol. 
SECTION: Cell lysis, protein precipitation, and lysate quantitation
12. After collection of colorimetric data for BCA assay on a 96-well-plate reader, create a standard curve, and fit the average of the triplicate values for each sample dilution to the standard curve to determine the protein concentration of each sample, after correcting for dilutions.
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
20. Separate the proteins in each fraction by MW using SDS–PAGE, being careful not to run lower-MW proteins off the gel that may be of interest for TDMS or TDP characterization. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
21. Remove the gel and submerge it in fixing solution in a clean container. Place on an orbital shaker for 20 min at low speed to fix the gel
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
22. Replace the fixing solution with enough washing solution to submerge the gel. Shake for 10 min at RT. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
23. Replace the washing solution with sensitizing solution. Shake for 1 min at low speed to sensitize the gel. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
24. Pour out the sensitizing solution and rinse the gel with two washes of dH2O for 1 min each. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
25. Add enough silver nitrate solution to submerge the gel and incubate it for 20 min on an orbital shaker at low speed to stain the gel. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
26. Pour out the silver nitrate solution and rinse the gel twice in dH2O for 1 min. 
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
27. Add enough developing solution to submerge the gel, and develop under rapid shaking. Discard the developing solution and replace it with fresh developing solution, once the liquid turns yellow, to reduce background staining.
SECTION: Desalting and concentrating GELFrEE fractions
29. To each fraction, add 4 volumes of Optima-grade methanol (600 µL), and vortex each tube vigorously for 20 s.
SECTION: Quality control of GELFrEE fraction by SDS–PAGE with silver staining
28. Pour out the developing solution and replace with terminating solution, once the desired staining intensity has been achieved, to terminate the development . The gel can now be scanned or photographed.
SECTION: Desalting and concentrating GELFrEE fractions
30. Add 1 volume (150 µL) of chloroform to each fraction, and vortex vigorously for 20 s. 
SECTION: Desalting and concentrating GELFrEE fractions
31. Add 3 volumes (450 µL) of LC–MS-grade water to each fraction. The liquid should immediately become cloudy and white in color. Vortex vigorously for 20 s
SECTION: Desalting and concentrating GELFrEE fractions
32. Centrifuge the tubes at 21,000g for 10 min at 4 °C. 
SECTION: Desalting and concentrating GELFrEE fractions
33. Carefully remove the tubes from the centrifuge. The separated aqueous and organic phases should be clearly observed, with a visible white protein pellet floating at the interface. Be very careful when transporting tubes to avoid shaking them or otherwise disturbing the protein pellet more than is necessary.
SECTION: Desalting and concentrating GELFrEE fractions
34. Remove and discard the top layer of solution, using extreme care to not remove or disturb the protein pellet. It may be necessary to leave a few millimeters of volume above the pellet to avoid accidentally pipetting it out. 
SECTION: Desalting and concentrating GELFrEE fractions
35. Very slowly add 3 volumes (450 µL) of Optima methanol to the tube, to avoid breaking up the protein pellet more than is necessary. Slowly mix the solution by carefully rocking the tube back and forth three to five times, while keeping an eye on the protein pellet and being careful to break it up as little as possible. 
SECTION: Desalting and concentrating GELFrEE fractions
36. Centrifuge the tubes at 21,000g for 10 min at 4 °C
SECTION: Desalting and concentrating GELFrEE fractions
37. At this point, the protein pellet should now be at the bottom of the tube. Carefully remove and discard as much methanol as possible from the tube without disturbing the pellet, and repeat steps 32–34 two more times. 
SECTION: Desalting and concentrating GELFrEE fractions
38. After the final methanol wash, remove as much methanol as possible from the tube without accidentally removing protein pellet particles. Place open tubes in a tube rack in an operating biological safety cabinet for 5–10 min to evaporate off any excess methanol. 
SECTION: Desalting and concentrating GELFrEE fractions
39. If sample is to be analyzed by CZE-MS/MS, pellet is resuspended in 10 uL 0.3% acetic acid. If sample is to be analyzed by LC-MS/MS, pellet is resuspended in 25 uL of buffer A (5% acetonitrile, 94.8% water, 0.2% formic acid).
SECTION: Desalting and concentrating GELFrEE fractions
40. Centrifuge the samples at 21,000g for 5 min at 4 °C, and slowly remove the majority of the solution, pipetting from just below the surface to avoid disturbing any unnoticed particles. Transfer each sample to a separate, pre-chilled LC vial. 
SECTION: Desalting and concentrating GELFrEE fractions
41. Transport the vials on ice to the nano-UHPLC auto sampler and analyze immediately to prevent sample degradation.
SECTION: Cell lysis, protein precipitation, and lysate quantitation
3. Sonicate lysate with a probe sonicator on ice (40% power, cycle 2 sec on, 3 sec off, 30 sec total).
 

SECTION: Cell lysis, protein precipitation, and lysate quantitation
4. Transfer lysate to 1.5 mL Lo-bind Eppendorf tube.
  

SECTION: Cell lysis, protein precipitation, and lysate quantitation
1.1. Fill liquid nitrogen dewar and equilibrate to 1.3 bar
SECTION: Cell lysis, protein precipitation, and lysate quantitation
1.2. Add frozen tissue sample to 25 mL grinding jar containing a 1-inch stainless steel ball.
SECTION: Cell lysis, protein precipitation, and lysate quantitation
1.3. Pulverize with three cycles of precooling with liquid nitrogen at 1 Hz for 3 min and grinding at 30 Hz for 1 min