3 Methods
All steps are performed at room temperature unless indicated
otherwise.
3.1 Preparation
of Protein Extracts
1. Homogenize 200 mg tissue in 25 volumes (5 mL) (see Notes
32) of PB buffer, pH 6.0 with protease inhibitors (see Note 33)
in thick wall ultracentrifuge tubes (see Notes 34). For biofluid
extracts, proceed directly to protein quantification (step 7).
2. Centrifuge at 100,000 Â g for 1 h at 4
C.
3. Transfer supernatant to fresh tubes and keep on ice.
4. Add 10 volumes (2 mL) of 1Â TBhS buffer, pH 7.2 with
protease inhibitors to the remaining pellets and homogenize
on ice.
5. Spin down at 16,000 Â g for 15 min at 4
C.
6. Transfer new supernatant to fresh tubes and keep on ice.
7. Proceed to protein quantification following quantification kit
instructions. Then, divide both supernatant (LIS/cytosolic/
sarcoplasmic fraction) and pellet (HIS/myofibrillar fraction)
fractions into aliquots (see Note 35). Take one and proceed
to the next step. Stored the rest at À80
C.
3.2 FTSC-Labeling
of Protein-Bound
Carbonyls
1. Adjust protein concentration at 1 mg/mL by adding PB buffer,
pH 6.0 to supernatant fraction and TBhS buffer, pH 7.2 to
resuspended pellet fraction (see Note 36).
2. Add 250 mM EDTA stock solution to 2.5 mM final concentration and mix.
3. Add 100 mM FTSC stock solution to 1 mM final concentration and mix. Protect samples from light by covering with
aluminum foil.
4. Incubate for 2.5 h at 37
C in the dark (see Note 37).
5. After incubation, add an equal volume of 20% TCA to the
samples in order to precipitate the protein (see Note 38).
6. Incubate for at least 20–30 min on ice (see Note 39).
7. Spin down at 16,000 Â g for 10 min at 4
C.
8. Discard the supernatant and carefully remove TCA in pellet by
adding 300 μL of 1:1 ethanol/ethyl acetate (see Note 40).
9. Vortex thoughtfully to break up the protein pellet (see Note 41).
10. Spin down at 16,000 Â g for 10 min at 4
C.
11. Carefully remove first 300 μL of 1:1 ethanol/ethyl
acetate wash.
12. Similarly carry out four more washes with 300 μL of 1:1
ethanol/ethyl acetate each (see Note 42).
Fluorescent 2DE-LC-MS for Carbonylome Analysis
235
All steps are performed at room temperature unless indicated
otherwise.
3.1 Preparation
of Protein Extracts
1. Homogenize 200 mg tissue in 25 volumes (5 mL) (see Notes
32) of PB buffer, pH 6.0 with protease inhibitors (see Note 33)
in thick wall ultracentrifuge tubes (see Notes 34). For biofluid
extracts, proceed directly to protein quantification (step 7).
2. Centrifuge at 100,000 Â g for 1 h at 4
C.
3. Transfer supernatant to fresh tubes and keep on ice.
4. Add 10 volumes (2 mL) of 1Â TBhS buffer, pH 7.2 with
protease inhibitors to the remaining pellets and homogenize
on ice.
5. Spin down at 16,000 Â g for 15 min at 4
C.
6. Transfer new supernatant to fresh tubes and keep on ice.
7. Proceed to protein quantification following quantification kit
instructions. Then, divide both supernatant (LIS/cytosolic/
sarcoplasmic fraction) and pellet (HIS/myofibrillar fraction)
fractions into aliquots (see Note 35). Take one and proceed
to the next step. Stored the rest at À80
C.
3.2 FTSC-Labeling
of Protein-Bound
Carbonyls
1. Adjust protein concentration at 1 mg/mL by adding PB buffer,
pH 6.0 to supernatant fraction and TBhS buffer, pH 7.2 to
resuspended pellet fraction (see Note 36).
2. Add 250 mM EDTA stock solution to 2.5 mM final concentration and mix.
3. Add 100 mM FTSC stock solution to 1 mM final concentration and mix. Protect samples from light by covering with
aluminum foil.
4. Incubate for 2.5 h at 37
C in the dark (see Note 37).
5. After incubation, add an equal volume of 20% TCA to the
samples in order to precipitate the protein (see Note 38).
6. Incubate for at least 20–30 min on ice (see Note 39).
7. Spin down at 16,000 Â g for 10 min at 4
C.
8. Discard the supernatant and carefully remove TCA in pellet by
adding 300 μL of 1:1 ethanol/ethyl acetate (see Note 40).
9. Vortex thoughtfully to break up the protein pellet (see Note 41).
10. Spin down at 16,000 Â g for 10 min at 4
C.
11. Carefully remove first 300 μL of 1:1 ethanol/ethyl
acetate wash.
12. Similarly carry out four more washes with 300 μL of 1:1
ethanol/ethyl acetate each (see Note 42).
Fluorescent 2DE-LC-MS for Carbonylome Analysis
235