TITLE: PURIFICATION OF PROTEINS FROM PFA FIXED SAMPLES BAK_WITH BIOTIN PULLDOWN FOR LC-MS/MS_2023
AUTHORS: Bryan Killinger

[DESCRIPTION]
This protocol details the purification of proteins from PFA fixed samples and extraction of proteins from formalin-fixed tissues. Also included, biotin pulldown prior to LC-MS/MS. Samples generated for this protocol have been used for mass spectrometry, immunoblotting, and pulldowns.

[STEPS]
SECTION: Procedure Day 1 (Extract proteins and test concentration)
1. Wash sections in DM 3 X 10 min.
SECTION: Procedure Day 1 (Extract proteins and test concentration)
2. Place sections in 1.5mL Eppendorf tube.
SECTION: Procedure Day 1 (Extract proteins and test concentration)
3. Add 0.5 mL of reversal buffer.

SECTION: Procedure Day 1 (Extract proteins and test concentration)
4. Briefly sonicate on low power to disperse tissue.
SECTION: Procedure Day 1 (Extract proteins and test concentration)
5. Add 5 µL of 100 millimolar (mM) PMSF. Mix well. Quick spin.

SECTION: Procedure Day 1 (Extract proteins and test concentration)
6. Heat on block for 98 °C for 30 min with cap locks.

SECTION: Procedure Day 1 (Extract proteins and test concentration)
1.1. Wash sections in DM for 10 min (1/3)

SECTION: Procedure Day 1 (Extract proteins and test concentration)
1.2. Wash sections in DM for 10 min (2/3)

SECTION: Procedure Day 1 (Extract proteins and test concentration)
1.3. Wash sections in DM for 10 min (3/3)
SECTION: Procedure Day 1 (Extract proteins and test concentration)
7. Remove from heat block carefully (caps will pop if not careful).
SECTION: Procedure Day 1 (Extract proteins and test concentration)
8.  After 5 min of cooling, vortex well. 

SECTION: Procedure Day 1 (Extract proteins and test concentration)
9. Centrifuge at 22000 x g for 30 min Room temperature.

SECTION: Procedure Day 1 (Extract proteins and test concentration)
10. Collect S1 (extracted proteins).
SECTION: Western blot (Optional)
11. Perform methanol/chloroform cleanup on 100 µL of S1.

SECTION: Western blot (Optional)
12. Resuspend resulting pellet in 100 µL 5% SDS.

SECTION: Western blot (Optional)
13. Perform BCA assay on 100 µL. Each BCA test takes 20 µL. The remaining volume can be used for western blot. Alternatively, more protein can be prepared from S1 if needed.

SECTION: Western blot (Optional)
14. Calculate volume required for 20 µg protein.

SECTION: Western blot (Optional)
15. 20ug / concentration of S1 ug/ul = ul of sample required 
SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
16. Add S1 to 10 mL TBST in 15mL conical tube. Mix well.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
17. Add 40 µL of prepared magnetic streptavidin beads to each tube.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
18. Nutation for 60 min Room temperature.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
19. Place tube on magnetic stand for 1 min (until all beads have been drawn out of solution).

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
20. Carefully remove supernatant no to disturb beads. Add 10 mL wash buffer.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
21. Nutation for 30 min.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
22. Place tube on magnetic stand for 1 min (until all beads have been drawn out of solution).

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
23. Add 10 mL wash buffer.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
24. Nutation for 30 min.

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
25. Place tube on magnetic stand for 1 min (until all beads have been drawn out of solution).

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
26. Add 10 mL wash buffer

SECTION: Procedure Day 2 (Capture biotinylated proteins and wash)
27. Nutation 1 min at 4 °C.

SECTION: Day 3 (Elute captured proteins)
28. Place tube on magnetic stand for 1 min (until all beads have been drawn out of solution).

SECTION: Day 3 (Elute captured proteins)
29. Carefully remove supernatant no to disturb beads.
SECTION: Day 3 (Elute captured proteins)
30. Add 1 mL of wash buffer.

SECTION: Day 3 (Elute captured proteins)
31. Mix samples until beads are suspended in wash buffer.
SECTION: Day 3 (Elute captured proteins)
32. Using a pipette with tip cut off, transfer suspended beads to a 1.5mL protein low bind tube. 
SECTION: Day 3 (Elute captured proteins)
33. Place tube on magnetic stand for 1 min (until all beads have been drawn out of solution).

SECTION: Day 3 (Elute captured proteins)
34. Remove buffer and surface wash with milliq 2X.
SECTION: Day 3 (Elute captured proteins)
35. Quick spin and remove liquid from tube (should only have beads left).
SECTION: Day 3 (Elute captured proteins)
36. Add 80 µL 1X SDS-page sample buffer containing reducing agent.

SECTION: Day 3 (Elute captured proteins)
37. Mix well and quick spin.
SECTION: Day 3 (Elute captured proteins)
38. Place on heat block set to 98 °C for 10 min.

SECTION: Day 3 (Elute captured proteins)
39. Mix well and quick spin.
SECTION: Day 3 (Elute captured proteins)
40. Place on magnet.
SECTION: Day 3 (Elute captured proteins)
41. Transfer eluent to two separate lo-bind tube and discard used beads. 35 µL in 1 tube (used for premass spec QC), the remaining 45 µL in another (used for mass spec).

SECTION: Day 3 (Elute captured proteins)
42. Immediately place in -80 °C for long-term storage.

SECTION: Day 4 (Prepare proteins for Mass Spec)
43. Prepare Bis-Tris 4-12% wedge gel 15 well.
SECTION: Day 4 (Prepare proteins for Mass Spec)
44. Make 800 mL 1X MOPS running buffer.
SECTION: Day 4 (Prepare proteins for Mass Spec)
45. Load 5 µL of MW standard in one side of gel.
SECTION: Day 4 (Prepare proteins for Mass Spec)
46. For MS load exactly 40 µL of the samples into wells. Careful not to spill into opposing wells. Only load samples into every other well to prevent cross contamination. 
SECTION: Day 4 (Prepare proteins for Mass Spec)
47. Ensure all wells have a buffer. For empty wells fill with 40 µL 1X loading buffer.
SECTION: Day 4 (Prepare proteins for Mass Spec)
48. Run the gel at 150V for a few minutes. Watch carefully until the sample has completely entered gel.
SECTION: Day 4 (Prepare proteins for Mass Spec)
49. Stop running, remove gel with clean gloves, and clean equipment.
SECTION: Day 4 (Prepare proteins for Mass Spec)
50. Fix gel in 100mL fixation solution (refer materials section) for 60 min Room temperature.

SECTION: Day 4 (Prepare proteins for Mass Spec)
51. Wash gel in several changes of milliq, until gel has swollen to original size.
SECTION: Day 4 (Prepare proteins for Mass Spec)
52. Place in 100 mL colloidal Coomassie blue stain solution. Cover loosely, and heat in microwave for few minutes until solution just starts to boil.

SECTION: Day 4 (Prepare proteins for Mass Spec)
53. Incubate gel in heated colloidal Coomassie blue stain solution for 8 min.

SECTION: Day 4 (Prepare proteins for Mass Spec)
54. Remove gel from stain solution and wash several times with milliq water. Clear background is achieved with ~120 min of washing.

SECTION: Day 4 (Prepare proteins for Mass Spec)
55. Using a clean razor excise the entire sample from the gel.
SECTION: Day 4 (Prepare proteins for Mass Spec)
56. Place each sample into a clean 1.5mL tube. Add 500 µL of milliq water.

SECTION: Day 4 (Prepare proteins for Mass Spec)
57. Samples can be stored at 4 °C until submitted to mass spec core.

SECTION: Optional: Pre mass spec QC
58. Estimating capture concentration - Purpose: To provide mass spec core with a rough estimate of the amount of protein in the capture sample. 

 


SECTION: Optional: Pre mass spec QC
58.1. Prepare gel as described above (Day 4, Steps 43-45)
SECTION: Optional: Pre mass spec QC
59. Load 20 µL of QC aliquot into wells.

SECTION: Optional: Pre mass spec QC
60. Load 2 µg BSA into 1 well.

SECTION: Optional: Pre mass spec QC
61. Run the gel at 150V for approximately 45 min or until the die has reached end of gel.

SECTION: Optional: Pre mass spec QC
62. Stain gel with Coomassie as described above (Day 4, Steps 50-54)
SECTION: Optional: Pre mass spec QC
63. Image on Odyssey using NIR.
SECTION: Optional: Pre mass spec QC
64. Forward image to NW mass spectrometry core.

Example of results; order = 2ug BSA, 3 captures, 2ug BSA, 3 captures, 2ug BSA, 3 captures
 

SECTION: Dot blot to estimate target enrichment
65. Cut PVDF membrane into ~1X3 inch piece. 
SECTION: Dot blot to estimate target enrichment
66. Activate PVDF in 100% methanol for 30 s

SECTION: Dot blot to estimate target enrichment
67. Equilibrate activated PVDF in diH20.
SECTION: Dot blot to estimate target enrichment
68. Just prior to blotting samples, place hydrated PVDF on wypall and quickly pat dry.
SECTION: Dot blot to estimate target enrichment
69. Place PVDF on new dry wypall.
SECTION: Dot blot to estimate target enrichment
70. Spot 1 µL of each sample onto PVDF.

SECTION: Dot blot to estimate target enrichment
71. Allow PVDF to dry completely. (~1h or 30 s)

SECTION: Dot blot to estimate target enrichment
72. Reactivate spotted PVDF as described above and equilibrate in dH20.
SECTION: Dot blot to estimate target enrichment
73. Blot can now be probed with antibodies using standard protocols.