23. Wash gel at least 3Â in destaining solution for 15 min (see
Note 61).
24. Capture Coomassie image under visible light.
25. Use PDQuest software or similar to analyze 2D gel images,
detected carbonylated spots and quantify specific protein carbonylation (see Note 62).
3.5 In-Gel Protein
Digestion
1. Cut spots from the gel and placed in 1.5 mL Eppendorf tube
(see Note 63).
2. Add 500 μL LC–MS H 2 O and shake at 1400 rpm for 5 min.
Repeat step 2 three times.
3. Remove the H 2 O and add 100 μL of 100% LC–MS AcN.
Vortex and shake at 1400 rpm for 15 min. Remove the AcN
and add 100 μL of LC–MS H 2 O. Vortex and shake at
1400 rpm for 5 min. Repeat step 3 twice.
4. Remove the H 2 O and add 100 μL of 100% LC–MS AcN.
Vortex and shake at 1400 rpm for 15 min.
5. Remove AcN with a tip and drying for ~5 min in a vacuum
dryer.
6. Star the digestion or store the samples at 4
C.
7. If samples were stored at 4
C overnight, leave them 20 min at
room temperature.
8. Add 20 μL of 25 ng/μL of trypsin solution per tube and let it
soak on ice for 30–45 min (see Note 64).
9. Remove the trypsin excess.
10. Add on top 30–50 μL of 25 mM ABC and ensure that the pH is
8.0 (see Note 65).
11. Incubate overnight at 37
C while gently shaking (300 rpm).
12. Centrifuge at 1000 Â g for 1 min. Collect supernatant in a
clean Eppendorf tube A.
13. Add 40 μL of 0.5% TFA, 50% AcN solution to each spot gel.
Vortex and shake at 1400 rpm for 20 min.
14. Centrifuge at 1000 Â g for 1 min. Collect supernatant in the
same Eppendorf tube A and vortex.
15. One spin and dry the mixture in the vacuum dryer.
16. Redissolve samples in 12 μL of 1% FA. Sonicate 5 min in an
ultrasonic bath (see Note 66).
17. Cleanup the protein digest before the LC–MS/MS analysis
using ZipTips or equivalent. To conditionate the column,
attach the ZipTip to a micropipette and wet the tip with 3Â
10 μL of 100% LC–MS methanol. Equilibrate then with 5Â
10 μL of 0.1% FA, 2% AcN solution. Loading 10 μL sample (see
Note 67). Wash with 5Â 10 μL of 0.1% FA, 2% AcN solution.
Elution with 10 μL of 0.1% FA, 50% AcN solution (see
Note 68).
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Lucı ´a Me ´ ndez et al.
Note 61).
24. Capture Coomassie image under visible light.
25. Use PDQuest software or similar to analyze 2D gel images,
detected carbonylated spots and quantify specific protein carbonylation (see Note 62).
3.5 In-Gel Protein
Digestion
1. Cut spots from the gel and placed in 1.5 mL Eppendorf tube
(see Note 63).
2. Add 500 μL LC–MS H 2 O and shake at 1400 rpm for 5 min.
Repeat step 2 three times.
3. Remove the H 2 O and add 100 μL of 100% LC–MS AcN.
Vortex and shake at 1400 rpm for 15 min. Remove the AcN
and add 100 μL of LC–MS H 2 O. Vortex and shake at
1400 rpm for 5 min. Repeat step 3 twice.
4. Remove the H 2 O and add 100 μL of 100% LC–MS AcN.
Vortex and shake at 1400 rpm for 15 min.
5. Remove AcN with a tip and drying for ~5 min in a vacuum
dryer.
6. Star the digestion or store the samples at 4
C.
7. If samples were stored at 4
C overnight, leave them 20 min at
room temperature.
8. Add 20 μL of 25 ng/μL of trypsin solution per tube and let it
soak on ice for 30–45 min (see Note 64).
9. Remove the trypsin excess.
10. Add on top 30–50 μL of 25 mM ABC and ensure that the pH is
8.0 (see Note 65).
11. Incubate overnight at 37
C while gently shaking (300 rpm).
12. Centrifuge at 1000 Â g for 1 min. Collect supernatant in a
clean Eppendorf tube A.
13. Add 40 μL of 0.5% TFA, 50% AcN solution to each spot gel.
Vortex and shake at 1400 rpm for 20 min.
14. Centrifuge at 1000 Â g for 1 min. Collect supernatant in the
same Eppendorf tube A and vortex.
15. One spin and dry the mixture in the vacuum dryer.
16. Redissolve samples in 12 μL of 1% FA. Sonicate 5 min in an
ultrasonic bath (see Note 66).
17. Cleanup the protein digest before the LC–MS/MS analysis
using ZipTips or equivalent. To conditionate the column,
attach the ZipTip to a micropipette and wet the tip with 3Â
10 μL of 100% LC–MS methanol. Equilibrate then with 5Â
10 μL of 0.1% FA, 2% AcN solution. Loading 10 μL sample (see
Note 67). Wash with 5Â 10 μL of 0.1% FA, 2% AcN solution.
Elution with 10 μL of 0.1% FA, 50% AcN solution (see
Note 68).
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Lucı ´a Me ´ ndez et al.