2.4 Protein
Extraction and
Quantification
1. RIPA buffer (150 mM sodium chloride (NaCl), 20 mM Tris–
HCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS)).
2. Protease inhibitor cocktail.
3. Centrifuge for 1.5-mL tubes.
4. Acetone.
5. Pierce Rapid Gold BCA Protein Assay Kit.
6. Eppendorf
® Thermomixer
® .
7. Sonicator.
2.5 Protein Digestion
and TMT Labeling
1. 1 M triethylammonium bicarbonate, stock solution (TEAB
buffer) (C 7 H 17 NO 3 ).
2. 200 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (C 9 H 15 O 6 P)) in 0.2 M TEAB.
3. 375 mM iodoacetamide (IAA) (C 2 H 4 INO): 1.5 mg
IAA + 74 μL of 0.1 M TEAB.
4. Pierce Trypsin Protease MS grade.
5. Culture centrifuge.
6. TMT10plex Mass Tag Labeling Kit.
7. Eppendorf
® Thermomixer
® .
8. Advanced Vortex Mixer ZX3.
9. Acetonitrile (AcN) (CH 3 CN).
10. Hydroxylamine (HXAM) (H 3 NO).
2.6 Peptide BasicReversed-Phase
Fractionation
1. High pH Reversed-Phase Peptide Fractionation Kit including
0.1% triethylamine (C 6 H 15 N) and spin columns.
2. 0.1% TFA in water.
3. Sonicator.
4. Speed-vac.
2.7 Liquid
ChromatographyTandem Mass
Spectrometry (LC-MS/
MS)
1. Nano-HPLC Bidimensional Dionex UltiMate 3000.
2. The
high-resolution
mass
spectrometer
Thermo
Q-Exactive HF.
3. The Proteome Discoverer 2.4 search engine.
3 Methods
3.1 EV Extraction
from Culture Medium
by Serial
Ultracentrifugation
1. Three days before starting the ultracentrifugation process of
EV extraction from MSCs in culture, the regular fetal bovine
serum is replaced by exosome-depleted fetal bovine serum to
guarantee that the EVs do not originate from the fetal bovine
serum.
Extracellular Vesicles from Mesenchymal Stem Cells
5
Extraction and
Quantification
1. RIPA buffer (150 mM sodium chloride (NaCl), 20 mM Tris–
HCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS)).
2. Protease inhibitor cocktail.
3. Centrifuge for 1.5-mL tubes.
4. Acetone.
5. Pierce Rapid Gold BCA Protein Assay Kit.
6. Eppendorf
® Thermomixer
® .
7. Sonicator.
2.5 Protein Digestion
and TMT Labeling
1. 1 M triethylammonium bicarbonate, stock solution (TEAB
buffer) (C 7 H 17 NO 3 ).
2. 200 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (C 9 H 15 O 6 P)) in 0.2 M TEAB.
3. 375 mM iodoacetamide (IAA) (C 2 H 4 INO): 1.5 mg
IAA + 74 μL of 0.1 M TEAB.
4. Pierce Trypsin Protease MS grade.
5. Culture centrifuge.
6. TMT10plex Mass Tag Labeling Kit.
7. Eppendorf
® Thermomixer
® .
8. Advanced Vortex Mixer ZX3.
9. Acetonitrile (AcN) (CH 3 CN).
10. Hydroxylamine (HXAM) (H 3 NO).
2.6 Peptide BasicReversed-Phase
Fractionation
1. High pH Reversed-Phase Peptide Fractionation Kit including
0.1% triethylamine (C 6 H 15 N) and spin columns.
2. 0.1% TFA in water.
3. Sonicator.
4. Speed-vac.
2.7 Liquid
ChromatographyTandem Mass
Spectrometry (LC-MS/
MS)
1. Nano-HPLC Bidimensional Dionex UltiMate 3000.
2. The
high-resolution
mass
spectrometer
Thermo
Q-Exactive HF.
3. The Proteome Discoverer 2.4 search engine.
3 Methods
3.1 EV Extraction
from Culture Medium
by Serial
Ultracentrifugation
1. Three days before starting the ultracentrifugation process of
EV extraction from MSCs in culture, the regular fetal bovine
serum is replaced by exosome-depleted fetal bovine serum to
guarantee that the EVs do not originate from the fetal bovine
serum.
Extracellular Vesicles from Mesenchymal Stem Cells
5