9. Vacuum dryer.
10. ZipTip
® Pipette Tips or similar 10 μL pipette tips with a 0.6 μL
bed of C18 resin.
11. 5% Formic acid (FA) solution: add 50 μL of LC–MS FA to
995 μL LC–MS H 2 O.
12. Equilibration solution for ZipTips: 0.1% FA, 2% AcN solution.
Add 50 μL of LC–MS FA to 995 μL LC–MS H 2 O.
13. Binding solution for ZipTips: 0.1% FA, 2% AcN solution. Add
50 μL of LC–MS FA to 950 μL LC–MS H 2 O.
14. Elution solution for ZipTips: 0.1% FA, 50% AcN solution. Add
500 μL of LC–MS AcN, 100 μL of 1% FA solution, and 400 μL
of LC–MS H 2 O.
15. 1% FA: add 10 μL of LC–MS FA to 990 μL LC–MS H 2 O.
2.6 LC–MS/MS
Analysis
of Carbonylome
1. Loading buffer: 0.1% FA, 1% AcN in LC–MS H 2 O. Add about
990 mL H 2 O to a 1 L graduated cylinder, and make up to 1 L
with AcN. Transfer to a bottle, and add 1 mL of FA.
2. Mobile phase A: 0.1% FA in LC–MS H 2 O. Add 100 μL FA to
100 mL of LC–MS H 2 O.
3. Mobile phase B: 0.1% FA in 100% AcN. Add 100 μL FA to
100 mL of LC–MS AcN.
4. Sonicate to degas the solutions for 15 min in an ultrasonic bath.
5. Prepare a stock solution of 8 pmol/μL Cytochrome C standard
digest: reconstituted the 1.6 nmol lyophilized cytochrome C
digest in 200 μL 0.1% FA, 2% AcN. Vortex briefly and wait at
least 10 min to ensure reconstitution of all peptides. Dilute the
stock solution to 500 fmol/μL as follows: prepare 150 μL
loading buffer in an autosampler vial (with insert) and add
10 μL from the 8 pmol/μL cytochrome C standard digest
stock solution and mix (with pipette or by vortexing) briefly
to homogenate the solution. Ensure there are no air bubbles at
the bottom of the vial.
6. Acclaim
® PepMap™ μ-Precolumn (0.3 Â 5 mm) or equivalent
trap column.
7. Acclaim
® PepMap™ RSLC C18 (2 μm, 100 A ˚ , 75 μm
id  15 cm) or equivalent C18 column.
8. Dionex™ UltiMate 3000 Series or similar NanoLC system.
9. LTQ™ Velos Pro Velos Pro or similar mass spectrometer.
10. PEAKS DB or similar software for data analysis and protein
identification.
234
Lucı ´a Me ´ ndez et al.
10. ZipTip
® Pipette Tips or similar 10 μL pipette tips with a 0.6 μL
bed of C18 resin.
11. 5% Formic acid (FA) solution: add 50 μL of LC–MS FA to
995 μL LC–MS H 2 O.
12. Equilibration solution for ZipTips: 0.1% FA, 2% AcN solution.
Add 50 μL of LC–MS FA to 995 μL LC–MS H 2 O.
13. Binding solution for ZipTips: 0.1% FA, 2% AcN solution. Add
50 μL of LC–MS FA to 950 μL LC–MS H 2 O.
14. Elution solution for ZipTips: 0.1% FA, 50% AcN solution. Add
500 μL of LC–MS AcN, 100 μL of 1% FA solution, and 400 μL
of LC–MS H 2 O.
15. 1% FA: add 10 μL of LC–MS FA to 990 μL LC–MS H 2 O.
2.6 LC–MS/MS
Analysis
of Carbonylome
1. Loading buffer: 0.1% FA, 1% AcN in LC–MS H 2 O. Add about
990 mL H 2 O to a 1 L graduated cylinder, and make up to 1 L
with AcN. Transfer to a bottle, and add 1 mL of FA.
2. Mobile phase A: 0.1% FA in LC–MS H 2 O. Add 100 μL FA to
100 mL of LC–MS H 2 O.
3. Mobile phase B: 0.1% FA in 100% AcN. Add 100 μL FA to
100 mL of LC–MS AcN.
4. Sonicate to degas the solutions for 15 min in an ultrasonic bath.
5. Prepare a stock solution of 8 pmol/μL Cytochrome C standard
digest: reconstituted the 1.6 nmol lyophilized cytochrome C
digest in 200 μL 0.1% FA, 2% AcN. Vortex briefly and wait at
least 10 min to ensure reconstitution of all peptides. Dilute the
stock solution to 500 fmol/μL as follows: prepare 150 μL
loading buffer in an autosampler vial (with insert) and add
10 μL from the 8 pmol/μL cytochrome C standard digest
stock solution and mix (with pipette or by vortexing) briefly
to homogenate the solution. Ensure there are no air bubbles at
the bottom of the vial.
6. Acclaim
® PepMap™ μ-Precolumn (0.3 Â 5 mm) or equivalent
trap column.
7. Acclaim
® PepMap™ RSLC C18 (2 μm, 100 A ˚ , 75 μm
id  15 cm) or equivalent C18 column.
8. Dionex™ UltiMate 3000 Series or similar NanoLC system.
9. LTQ™ Velos Pro Velos Pro or similar mass spectrometer.
10. PEAKS DB or similar software for data analysis and protein
identification.
234
Lucı ´a Me ´ ndez et al.