2.4 Gel-Free
Proteomics:
Label-Free
1. Pierce 660 nm Protein Assay, plus the Ionic Detergent Compatibility Reagent (IDCR).
2. 10 kDa Amicon™ Ultra-0.5.
3. 0.1 M NaOH.
4. UA buffer: 8 M Urea and 0.1 M Tris Base (pH 8.5).
5. 25 mM TEAB.
6. DTT 1 M in 25 mM TEAB.
7. 0.05 M iodoacetamide (IAA) in UA.
8. 0.1μg/μL trypsin in 20 mM TEAB.
9. Savant™ SPD121P SpeedVac.
10. 5% methanol/0.5% TFA.
11. Solvent A: 0.1% formic acid.
12. Solvent B: acetonitrile 0.1% formic acid.
13. Agilent 1200 nanoflow system.
14. C18 preconcentration cartridge.
15. C18 column.
16. LTQ-Orbitrap XL mass spectrometer.
17. Nanoelectrospray ion source.
18. Progenesis QI software (v 4.0).
19. Proteome Discoverer v1.4.
20. SwissProt human database.
3 Methods
The workflow of the experimental approach is shown in Fig. 1.
3.1 Blood Collection
Blood collection should be performed as recommended by the
ISTH SSC collaborative workshop in order to minimize the impact
of pre-analytical parameters on the measurement of circulating
microparticles [10].
1. Collect 50 mL of blood in coagulation 3.2% sodium citrate
tubes (see Notes 1 and 2).
2. Add 450μL of warmed ACD to the blood and centrifuge it at
200 Â g for 20 min.
3. Pippete off the supernatant (platelet-rich plasma (PRP)) into a
50-mL centrifuge tube.
4. Add 10μg prostacyclin (10μL of stock solution at 1 mg/mL),
mix gently by inversion, and immediately centrifuge at
1000 Â g for 10 min (see Note 3).
5. Pippete off the supernatant (platelet-poor plasma (PPP)) into a
50-mL centrifuge tube. Centrifuge it at 1500 Â g for 10 min.
16
Maria N. Barrachina and A ´ ngel Garcı ´a
Proteomics:
Label-Free
1. Pierce 660 nm Protein Assay, plus the Ionic Detergent Compatibility Reagent (IDCR).
2. 10 kDa Amicon™ Ultra-0.5.
3. 0.1 M NaOH.
4. UA buffer: 8 M Urea and 0.1 M Tris Base (pH 8.5).
5. 25 mM TEAB.
6. DTT 1 M in 25 mM TEAB.
7. 0.05 M iodoacetamide (IAA) in UA.
8. 0.1μg/μL trypsin in 20 mM TEAB.
9. Savant™ SPD121P SpeedVac.
10. 5% methanol/0.5% TFA.
11. Solvent A: 0.1% formic acid.
12. Solvent B: acetonitrile 0.1% formic acid.
13. Agilent 1200 nanoflow system.
14. C18 preconcentration cartridge.
15. C18 column.
16. LTQ-Orbitrap XL mass spectrometer.
17. Nanoelectrospray ion source.
18. Progenesis QI software (v 4.0).
19. Proteome Discoverer v1.4.
20. SwissProt human database.
3 Methods
The workflow of the experimental approach is shown in Fig. 1.
3.1 Blood Collection
Blood collection should be performed as recommended by the
ISTH SSC collaborative workshop in order to minimize the impact
of pre-analytical parameters on the measurement of circulating
microparticles [10].
1. Collect 50 mL of blood in coagulation 3.2% sodium citrate
tubes (see Notes 1 and 2).
2. Add 450μL of warmed ACD to the blood and centrifuge it at
200 Â g for 20 min.
3. Pippete off the supernatant (platelet-rich plasma (PRP)) into a
50-mL centrifuge tube.
4. Add 10μg prostacyclin (10μL of stock solution at 1 mg/mL),
mix gently by inversion, and immediately centrifuge at
1000 Â g for 10 min (see Note 3).
5. Pippete off the supernatant (platelet-poor plasma (PPP)) into a
50-mL centrifuge tube. Centrifuge it at 1500 Â g for 10 min.
16
Maria N. Barrachina and A ´ ngel Garcı ´a