2.5 In-Gel Protein
Digestion
1. 2.5-mL LoBind tubes.
2. Bands wash solution: 50% (v/v) acetonitrile/25 mM ammonium bicarbonate.
3. Acetonitrile (ACN).
4. Trifluoroacetic acid (TFA).
5. Wash solution: 50% (v/v) ACN/25 mM Ambic.
6. Reduction solution: 10 mM DTT in 50 mM ammonium
bicarbonate.
7. Alkylation solution: 55 mM iodoacetamide in 50 mM ammonium bicarbonate.
8. Sequencing-grade trypsin (20 ng/μL in 12.5 mM Ambic/2%
(v/v) ACN).
9. 5% (v/v) formic acid in Milli-Q water.
10. Extracted solution: 50% (v/v) ACN/0.1% (v/v) TFA.
11. Eppendorf Thermo Mixer
®
.
12. SpeedVac™ concentrator.
2.6 SWATH-MS
Methods
1. Solution A (Milli-Q water + 0.1% formic acid) (see Note 1).
2. Solution B (ACN + 0.1% formic acid) for liquid chromatography (see Note 1).
3. TripleTOF
® 6600+.
4. Nano-LC and autosampler (Eksigent
®
425 nano-LC,
ekspert™ 400 autosampler).
5. C18 Trap YMC—200 Â 0.5 mm precolumn—3 μm 120 A ˚ .
6. Analytical ChromXP C18 LC columns Eksigent
®
.
7. SWATH-MS Acquisition Performance Kit, both PepCalMix
and K562 PROTEIN EXTRACT DIGEST.
8. Analyst TF 1.7.1.
9. PeakView 2.2 or higher.
10. Variable Window Calculator from Sciex.
11. Protein Pilot 5.0.2 or higher.
12. MS/MS (ALL) with SWATH-MS™ Acquisition MicroApp
2.0.1.2133.
13. Microsoft Excel.
14. MarkerView 1.3.1.
SWATH-MS Protocols in Human Diseases
113
Digestion
1. 2.5-mL LoBind tubes.
2. Bands wash solution: 50% (v/v) acetonitrile/25 mM ammonium bicarbonate.
3. Acetonitrile (ACN).
4. Trifluoroacetic acid (TFA).
5. Wash solution: 50% (v/v) ACN/25 mM Ambic.
6. Reduction solution: 10 mM DTT in 50 mM ammonium
bicarbonate.
7. Alkylation solution: 55 mM iodoacetamide in 50 mM ammonium bicarbonate.
8. Sequencing-grade trypsin (20 ng/μL in 12.5 mM Ambic/2%
(v/v) ACN).
9. 5% (v/v) formic acid in Milli-Q water.
10. Extracted solution: 50% (v/v) ACN/0.1% (v/v) TFA.
11. Eppendorf Thermo Mixer
®
.
12. SpeedVac™ concentrator.
2.6 SWATH-MS
Methods
1. Solution A (Milli-Q water + 0.1% formic acid) (see Note 1).
2. Solution B (ACN + 0.1% formic acid) for liquid chromatography (see Note 1).
3. TripleTOF
® 6600+.
4. Nano-LC and autosampler (Eksigent
®
425 nano-LC,
ekspert™ 400 autosampler).
5. C18 Trap YMC—200 Â 0.5 mm precolumn—3 μm 120 A ˚ .
6. Analytical ChromXP C18 LC columns Eksigent
®
.
7. SWATH-MS Acquisition Performance Kit, both PepCalMix
and K562 PROTEIN EXTRACT DIGEST.
8. Analyst TF 1.7.1.
9. PeakView 2.2 or higher.
10. Variable Window Calculator from Sciex.
11. Protein Pilot 5.0.2 or higher.
12. MS/MS (ALL) with SWATH-MS™ Acquisition MicroApp
2.0.1.2133.
13. Microsoft Excel.
14. MarkerView 1.3.1.
SWATH-MS Protocols in Human Diseases
113