TITLE: BAF_Protocol_007 Solution Digest with Protein Precipitation
AUTHORS: Nicholas Sherman

[DESCRIPTION]
The protocol is a method to digest a protein mixture obtained from a cell lysis or tissue that is in solution. The protocol includes a precipitation step to clean up the proteins before digestion. The protein mixture can then be digested and the resulting peptides cleaned up using protocol 003 and/or 006.

[BEFORE_START]
REAGENTS: (All reagents to be prepared fresh for each digestion)
1. 100 mM ammonium bicarbonate (ABC): 0.158 g in 20 mL distilled water
2. 50 mM ABC: 0.079g in 20 mL distilled water
3. Acetone
4. Methanol
5. 10% sodium dodecyl sulfate (SDS): 1g in 10 mL distilled water. 
6. 100 mM DTT: 0.0015g in 100 uLof 100mM ABC (DO NOT mix until directly before you are ready to use)
7. 500 mM Iodoacetamide: 0.01g in 100 uL of 100 mM ABC (DO NOT mix until directly before you are ready to use)
8. Trypsin solution: Keep on ice. Promega (cat. # V5113) is already diluted in 50 mM acetic acid.

[STEPS]
SECTION: Solution Tissue Digest with Precipitation
1. Add 5X  (volume to mg tissue) of lysis buffer (1% SDS, 100mM AmBiC) to tissue and transfer to Bead Mill 24 reinforced tube with 5 stainless steel balls.
SECTION: Solution Tissue Digest with Precipitation
2. Lyse the cells with Fisher Bead Mill 24 (speed: 5m/s, time: 20 sec, number of cycle: 3, dwell/pause between runs: 10 sec).
SECTION: Solution Tissue Digest with Precipitation
3. Centrifuge lysate at 16,000 x g for 10 min at 4°C.
SECTION: Solution Tissue Digest with Precipitation
4. Carefully separate the supernatant and transfer into a new 1.5mL Eppendorf tube.
SECTION: Solution Tissue Digest with Precipitation
13. Determine the protein concentration of the supernatant using BCA (5uL of 10X dilution)
SECTION: Solution Tissue Digest with Precipitation
14. Transfer 20ug into a new, pre-washed tube and adjust to a final volume of 100uL with 100mM AmBiC.
SECTION: Solution Tissue Digest with Precipitation
5. Add 5μL of 10 mM dithiothreitol (DTT) solution for 30 min at RT to reduce.
SECTION: Solution Tissue Digest with Precipitation
6. Add 5μL of 50 mM iodoacetamide (IA) solution for 30 min at RT to alkylate.
SECTION: Solution Tissue Digest with Precipitation
15. 12. Add 1 ug of modified trypsin solution overnight at 37°C.
SECTION: Solution Tissue Digest with Precipitation
16. 13. Add 8uL of formic acid to the sample to quench the reaction.
SECTION: Solution Tissue Digest with Precipitation
7. Transfer sample to 15 mL conical tube.
SECTION: Solution Tissue Digest with Precipitation
9. Centrifuge sample 1h at 4C at 3200 x g.
SECTION: Solution Tissue Digest with Precipitation
8. Perform precipitation by adding -20C acetone (1x volume of the sample) and -20C methanol (9 x volume of the sample) and incubate overnight at -80C.
SECTION: Solution Tissue Digest with Precipitation
10. Remove supernatant, wash pellet with 1 mL of -20C cold methanol and centrifuge.
SECTION: Solution Tissue Digest with Precipitation
11. Remove supernatant, air-dry pellet in the PCR workstation at RT till just damp.
SECTION: Solution Tissue Digest with Precipitation
12. Solubilize proteins in 50 uL (more depending on amount of tissue) of 50 mM Ammonium Bicarbonate
SECTION: Solution Tissue Digest with Precipitation
17. Speedvac to dry peptide sample.
SECTION: Solution Tissue Digest with Precipitation
18. Samples can be cleaned up using C18 (Protocol 003) and/or Magnetic beads (Protocol 006). 
SECTION: Cell Pellet Digest with Precipitation
25. Centrifuge samples 1h at 4C at 3200 xg
SECTION: Cell Pellet Digest with Precipitation
26. Remove supernatants, wash pellets with 1 mL of -20C cold methanol and centrifuge.
SECTION: Cell Pellet Digest with Precipitation
27. Remove supernatants, air-dry pellets in the PCR workstation at RT
SECTION: Cell Pellet Digest with Precipitation
28. Solubilize proteins in 50-100 uL of 50 mM Ammonium Bicarbonate (~1ug/uL concentration).
SECTION: Cell Pellet Digest with Precipitation
29. 11. Determine the protein concentration of the supernatant using BCA (use 5uL of 10X dilution sample).
SECTION: Cell Pellet Digest with Precipitation
30. 12. Transfer 20ug per condition into a pre-washed tube and adjust to a final volume of 100uL with 100mM AmBiC
SECTION: Cell Pellet Digest with Precipitation
31. 13. Add 1 ug of modified trypsin solution overnight at 37°C.
SECTION: Cell Pellet Digest with Precipitation
32. 14. Add 8uL of formic acid to the sample to quench the reaction.
SECTION: Cell Pellet Digest with Precipitation
33. 15. Speedvac to dry peptide sample.
SECTION: Cell Pellet Digest with Precipitation
19. Add lysis buffer to final concentration 1% SDS using 100mM AmBiC and protease inhibitor cocktail (1:100 dilution) in 1.5 mL tubes. This is generally 10E6 cells but can be adjusted for 10E4-10E7. 
SECTION: Cell Pellet Digest with Precipitation
20. Submit samples to 3 cycles at 90C and RT incubation as follows: shake at 800 rpm for 10 min at 90C in temperature controlled thermal shaker and incubate at RT for 10 min.
SECTION: Cell Pellet Digest with Precipitation
21. Add ~60 uL of 100 mM DDT (final conc. 10 mM) to samples and incubate for 30 min at RT
SECTION: Cell Pellet Digest with Precipitation
22. Add ~60 uL of 500 mM IA (final conc. 50 mM) to samples and incubate for 30 min at RT in the dark.
SECTION: Cell Pellet Digest with Precipitation
23. Transfer samples to 15 mL tubes (~700 uL).
SECTION: Cell Pellet Digest with Precipitation
24. Perform precipitation by adding 700 ul of -20C cold acetone (same volume of the sample) and 7200 mL of -20C cold methanol (9 volumes of the sample) and incubate overnight at -80C
SECTION: Cell Pellet Digest with Precipitation
34. Samples can be cleaned up using C18 (Protocol 003) and/or Magnetic beads (Protocol 006).