5. Orbital shaker.
6. Blue light transilluminator.
7. Scalpels.
8. Protein low bind microtubes.
2.4 In-Gel Protein
Digestion
1. Ultrapure (HPLC-grade) water.
2. HPLC-grade acetonitrile.
3. 25 mM ammonium bicarbonate.
4. 10 mM dithiothreitol (DTT) in 25 mM ammonium
bicarbonate.
5. 100 mM iodoacetamide (IAM) in 25 mM ammonium
bicarbonate.
6. Sequencing-grade modified trypsin (12.5 ng/μL, resuspended
in 25 mM ammonium bicarbonate).
7. 0.5% Formic acid solution.
8. 50% Acetonitrile/0.1% formic acid.
9. Ice.
10. Vortex.
11. Thermoblock.
12. Speed-vacuum.
13. Filter tips (optional).
14. pH strips.
15. Protein low bind microtubes.
2.5 IsobaricLabeling Using
TMT-Multiplex
1. HPLC-grade water.
2. HPLC-grade acetone.
3. 100 mM Triethylammonium bicarbonate (TEAB).
4. 200 mM Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP).
5. 375 mM IAM in 100 mM TEAB.
6. Sequencing-grade modified trypsin (20 μg of lyophilized
enzyme resuspended in 200 μL of 100 mM TEAB).
7. HPLC-grade acetonitrile.
8. TMT6plex™, TMT10plex™, or TMT11plex™ Mass Tag
Labeling Kit.
9. 5% Hydroxylamine.
10. Pierce High pH Reversed-Phase Peptide Fractionation Kit.
11. Refrigerated centrifuge.
12. Vortex.
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Angel P. Diz and Paula Sa ´ nchez-Marı ´n