6. Formic acid, LC-MS grade.
7. MS solvent: 3% acetonitrile, 0.1% formic acid in LC-MS grade
water.
2.5 In-Gel Digestion
for Bottom-up
Proteomics
1. Stained gel containing protein sample (see Notes 6 and 7).
2. Glass plate, cleaned with 70% v/v ethanol prior to use.
3. Razor blade, cleaned with 70% v/v ethanol prior to use.
4. 70% v/v Ethanol.
5. 37
C incubator.
6. Vacuum concentrator.
7. Acetonitrile, LC-MS Grade.
8. 0.2 M ammonium bicarbonate.
9. Formic acid, LC-MS grade.
10. Trifluoroacetic acid (TFA), high purity.
11. Destain solution: 60% v/v acetonitrile, in 0.2 M ammonium
bicarbonate.
12. Extraction solution: 60% v/v acetonitrile, 0.1% v/v TFA.
13. Modified trypsin sequencing grade: 0.1 mg/mL in 0.2 M
ammonium bicarbonate (see Notes 8 and 9).
14. Gel-loading pipette tips.
15. Water, LC-MS grade.
16. MS loading solvent: 3% v/v acetonitrile, 0.1% v/v formic acid
(see Note 10).
17. Deplasticized 0.65 mL microcentrifuge tubes: Microcentrifuge
tubes must be pretreated to remove plastic polymers prior to
use in mass spectrometry. Fill the tubes with extraction solution, invert tubes to mix, incubate at room temperature for 1 h,
and decant the solution. Repeat this process two times for each
tube. After the final wash, let tubes to air dry inside of a fume
hood or dry using a vacuum concentrator. Tubes must be
thoroughly dry prior to use.
2.6 In-Solution
Digestion
for Bottom-up
Proteomics
1. 30 μg of protein sample (see Note 11).
2. Deplasticized microcentrifuge tubes (see item 17 in
Subheading 2.5).
3. Water, LC-MS grade.
4. 8 M Urea: Urea should be deionized prior to use to remove
reactive cyanate ions. 8 M solutions of urea may be bulk deionized using an ion exchange resin. Add AG-501-X8 (Bio-Rad,
Hercules, CA) at 5% w/v, stir the solution for 2 h at RT, and
filter the solution to recover deionized 8 M urea.
5. 50 mM ammonium bicarbonate.
Proteomic Analyses
537
7. MS solvent: 3% acetonitrile, 0.1% formic acid in LC-MS grade
water.
2.5 In-Gel Digestion
for Bottom-up
Proteomics
1. Stained gel containing protein sample (see Notes 6 and 7).
2. Glass plate, cleaned with 70% v/v ethanol prior to use.
3. Razor blade, cleaned with 70% v/v ethanol prior to use.
4. 70% v/v Ethanol.
5. 37
C incubator.
6. Vacuum concentrator.
7. Acetonitrile, LC-MS Grade.
8. 0.2 M ammonium bicarbonate.
9. Formic acid, LC-MS grade.
10. Trifluoroacetic acid (TFA), high purity.
11. Destain solution: 60% v/v acetonitrile, in 0.2 M ammonium
bicarbonate.
12. Extraction solution: 60% v/v acetonitrile, 0.1% v/v TFA.
13. Modified trypsin sequencing grade: 0.1 mg/mL in 0.2 M
ammonium bicarbonate (see Notes 8 and 9).
14. Gel-loading pipette tips.
15. Water, LC-MS grade.
16. MS loading solvent: 3% v/v acetonitrile, 0.1% v/v formic acid
(see Note 10).
17. Deplasticized 0.65 mL microcentrifuge tubes: Microcentrifuge
tubes must be pretreated to remove plastic polymers prior to
use in mass spectrometry. Fill the tubes with extraction solution, invert tubes to mix, incubate at room temperature for 1 h,
and decant the solution. Repeat this process two times for each
tube. After the final wash, let tubes to air dry inside of a fume
hood or dry using a vacuum concentrator. Tubes must be
thoroughly dry prior to use.
2.6 In-Solution
Digestion
for Bottom-up
Proteomics
1. 30 μg of protein sample (see Note 11).
2. Deplasticized microcentrifuge tubes (see item 17 in
Subheading 2.5).
3. Water, LC-MS grade.
4. 8 M Urea: Urea should be deionized prior to use to remove
reactive cyanate ions. 8 M solutions of urea may be bulk deionized using an ion exchange resin. Add AG-501-X8 (Bio-Rad,
Hercules, CA) at 5% w/v, stir the solution for 2 h at RT, and
filter the solution to recover deionized 8 M urea.
5. 50 mM ammonium bicarbonate.
Proteomic Analyses
537