3. 100 mM fluorescein-5-thiosemicarbazide (FTSC, !99.0%) in
50% acetonitrile (AcN): dissolve 6.3 mg in 150 μL of 1:1
H 2 O/AcN solution (see Note 7).
4. 2D buffer (urea/thiourea lysis buffer): 7 M urea (!99.5%),
2 M thiourea (!99.0%), 2% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate hydrate (CHAPS, !98.0%),
0.4% dithiothreitol (DTT), 0.5% pharmalyte 3–10 carrier
ampholytes or similar (see Note 8) and 0.5% immobilized pH
gradient (IPG) buffer (see Note 9). Dissolve 22.0 g urea in
about 25 mL of deionized H 2 O with stirring. Then, slowly add
8.0 g thiourea (see Note 10) and adjust the volume to 50 mL
with deionized H 2 O. Add 0.5 g of Serdolit MB-1 mixed-bed
ion-exchange resin or similar, stir for 20 min, and filter (see
Note 11). To 48 mL of the solution add 1.0 g of CHAPS,
0.25 mL of pharmalyte pH 3–10, and 0.25 mL of IPG buffer,
and adjust the solution to a final volume of 50 mL using a
volumetric flask. Store 1 mL-aliquots of this urea/thiourea
solution at À80
C until required. Immediately before use,
add 0.2 g of DTT to the required aliquot volume and discard
the leftover solution.
5. 20% (w/v) Trichloroacetic acid (TCA): dissolve 20 g of TCA in
100 mL of deionized H 2 O (see Note 12). Store at 4
C
until use.
6. 1:1 Ethanol/ethyl acetate: prepare 1 L of 1:1 ethanol/ethyl
acetate by mixing 500 mL of ethanol absolute (!99.5%) and
500 mL ethyl acetate (!99.5%) (see Note 13).
7. Refrigerated microcentrifuge.
8. Ultrasonic bath.
9. Thermoblock.
10. Vacuum dryer.
11. Protein quantification Bradford kit (see Note 14).
12. Spectrophotometer capable of measuring absorbance in the
595 nm region.
2.3 First Dimension:
Isoelectrofocusing
(IEF)
1. Rehydration solution: 2D buffer (urea/thiourea lysis buffer)
(see Note 15).
2. Prepare 500 mL stock of 1.5 M Tris–HCl, pH 8.8. Weigh
90.86 g Tris–HCl and add about 450 mL of water. When
Tris is fully dissolved, adjust for pH with HCl (see Note 3).
Then, transfer to a 500 mL volumetric flask and complete with
H 2 O up to a volume of 500 mL and mix. Store at 4
C.
3. 10% (w/v) Sodium dodecyl sulfate (SDS) solution: prepare a
1 L stock of 10% SDS dissolved in H 2 O. Weigh 100 g SDS, add
H 2 O up to a volume of 1 L, and mix (see Note 16).
Fluorescent 2DE-LC-MS for Carbonylome Analysis
231
50% acetonitrile (AcN): dissolve 6.3 mg in 150 μL of 1:1
H 2 O/AcN solution (see Note 7).
4. 2D buffer (urea/thiourea lysis buffer): 7 M urea (!99.5%),
2 M thiourea (!99.0%), 2% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate hydrate (CHAPS, !98.0%),
0.4% dithiothreitol (DTT), 0.5% pharmalyte 3–10 carrier
ampholytes or similar (see Note 8) and 0.5% immobilized pH
gradient (IPG) buffer (see Note 9). Dissolve 22.0 g urea in
about 25 mL of deionized H 2 O with stirring. Then, slowly add
8.0 g thiourea (see Note 10) and adjust the volume to 50 mL
with deionized H 2 O. Add 0.5 g of Serdolit MB-1 mixed-bed
ion-exchange resin or similar, stir for 20 min, and filter (see
Note 11). To 48 mL of the solution add 1.0 g of CHAPS,
0.25 mL of pharmalyte pH 3–10, and 0.25 mL of IPG buffer,
and adjust the solution to a final volume of 50 mL using a
volumetric flask. Store 1 mL-aliquots of this urea/thiourea
solution at À80
C until required. Immediately before use,
add 0.2 g of DTT to the required aliquot volume and discard
the leftover solution.
5. 20% (w/v) Trichloroacetic acid (TCA): dissolve 20 g of TCA in
100 mL of deionized H 2 O (see Note 12). Store at 4
C
until use.
6. 1:1 Ethanol/ethyl acetate: prepare 1 L of 1:1 ethanol/ethyl
acetate by mixing 500 mL of ethanol absolute (!99.5%) and
500 mL ethyl acetate (!99.5%) (see Note 13).
7. Refrigerated microcentrifuge.
8. Ultrasonic bath.
9. Thermoblock.
10. Vacuum dryer.
11. Protein quantification Bradford kit (see Note 14).
12. Spectrophotometer capable of measuring absorbance in the
595 nm region.
2.3 First Dimension:
Isoelectrofocusing
(IEF)
1. Rehydration solution: 2D buffer (urea/thiourea lysis buffer)
(see Note 15).
2. Prepare 500 mL stock of 1.5 M Tris–HCl, pH 8.8. Weigh
90.86 g Tris–HCl and add about 450 mL of water. When
Tris is fully dissolved, adjust for pH with HCl (see Note 3).
Then, transfer to a 500 mL volumetric flask and complete with
H 2 O up to a volume of 500 mL and mix. Store at 4
C.
3. 10% (w/v) Sodium dodecyl sulfate (SDS) solution: prepare a
1 L stock of 10% SDS dissolved in H 2 O. Weigh 100 g SDS, add
H 2 O up to a volume of 1 L, and mix (see Note 16).
Fluorescent 2DE-LC-MS for Carbonylome Analysis
231