2.3 Protein
Concentration
1. Bicinchoninic acid (BCA) method.
2. Centrifuge.
3. Microplate reader (Multiskan™ GO, Thermo Fisher
Scientific).
2.4 Accelerated
Trypsin Protein
Digestion Using HIFU
1. 8 M Urea.
2. 100 mM DTT (DL-dithiothreitol) !98% (HPLC).
3. 500 mM IAA (iodoacetamide).
4. Sequencing-grade modified trypsin (Promega, Madison,
WI, USA).
5. 25 mM ammonium bicarbonate, pH 8.
6. High-intensity focused ultrasonic (HIFU) probe with a 1-mm
tip (Dr. Heilscher, Teltow, Germany).
7. Trifluoroacetic acid (TFA).
8. SepPak C18 cartridge from Waters (Milford, MA, USA).
9. SpeedVac concentrator.
2.5 Phosphopeptide
Enrichment Using TiO 2
1. Mobicol spin columns (1 mL).
2. TiO 2 packing resin (GL Science, Saitama, Japan).
3. Washing solution A: 80% acetonitrile (ACN) and 3.5% TFA
saturated with phthalic acid (100 mg phthalic acid/mL).
4. Methanol LC-MS grade.
5. Acetonitrile (ACN) LC-MS grade.
6. End-over-end rotator.
7. Washing solution B: 80% ACN and 0.1% TFA solution.
8. Washing solution C: 0.1% TFA.
9. Elution buffer: 0.3 M NH 4 OH (pH 10.5).
10. Trifluoroacetic acid (TFA) LC-MS grade.
11. C18 SepPak cartridge (Waters Corporation, Milford, MA).
2.6 Phosphopeptide
Fractionation Using
SCX
1. Poly SULFOETHYL -A SCX cartridge columns (The Nest
Group Inc. Southborough, MA).
2. Vacuum manifold.
3. Equilibration buffer: 30% ACN containing 5 mM KH 2 PO 4 ,
pH 2.7.
4. TFA.
5. 0.05% formic acid (FA).
6. 5 mM KH 2 PO 4 .
7. 30% ACN.
Rapid Shotgun Phosphoproteomics Analysis
261
Concentration
1. Bicinchoninic acid (BCA) method.
2. Centrifuge.
3. Microplate reader (Multiskan™ GO, Thermo Fisher
Scientific).
2.4 Accelerated
Trypsin Protein
Digestion Using HIFU
1. 8 M Urea.
2. 100 mM DTT (DL-dithiothreitol) !98% (HPLC).
3. 500 mM IAA (iodoacetamide).
4. Sequencing-grade modified trypsin (Promega, Madison,
WI, USA).
5. 25 mM ammonium bicarbonate, pH 8.
6. High-intensity focused ultrasonic (HIFU) probe with a 1-mm
tip (Dr. Heilscher, Teltow, Germany).
7. Trifluoroacetic acid (TFA).
8. SepPak C18 cartridge from Waters (Milford, MA, USA).
9. SpeedVac concentrator.
2.5 Phosphopeptide
Enrichment Using TiO 2
1. Mobicol spin columns (1 mL).
2. TiO 2 packing resin (GL Science, Saitama, Japan).
3. Washing solution A: 80% acetonitrile (ACN) and 3.5% TFA
saturated with phthalic acid (100 mg phthalic acid/mL).
4. Methanol LC-MS grade.
5. Acetonitrile (ACN) LC-MS grade.
6. End-over-end rotator.
7. Washing solution B: 80% ACN and 0.1% TFA solution.
8. Washing solution C: 0.1% TFA.
9. Elution buffer: 0.3 M NH 4 OH (pH 10.5).
10. Trifluoroacetic acid (TFA) LC-MS grade.
11. C18 SepPak cartridge (Waters Corporation, Milford, MA).
2.6 Phosphopeptide
Fractionation Using
SCX
1. Poly SULFOETHYL -A SCX cartridge columns (The Nest
Group Inc. Southborough, MA).
2. Vacuum manifold.
3. Equilibration buffer: 30% ACN containing 5 mM KH 2 PO 4 ,
pH 2.7.
4. TFA.
5. 0.05% formic acid (FA).
6. 5 mM KH 2 PO 4 .
7. 30% ACN.
Rapid Shotgun Phosphoproteomics Analysis
261