11. Add 41 μL of each vial to each corresponding sample and give
an average vortex once added to the samples. The leftover
reagent could be stored, tightly closed, at À20
C for future
experiments (see storage conditions in the manufacturer
brochure).
12. Incubate for 1 h at room temperature.
13. Just 5 min before the end of the incubation, prepare 5% HXAM
by adding 10 μL of 50% HXAM + 80 μL of H 2 O
(LC-MS) + 10 μL of 1 M TEAB. Vortex, spin, and then add
8 μL of 5% HXAM to the samples.
14. Incubate for 15 min at room temperature. Spin.
15. Transfer the entire contents of each labeling to the same 2-mL
vial. The final total volume should be ~1.7 mL containing a
total of 1000 μg of labeled peptides if all the channels have
been used and 100 μg of total protein per channel (see Note 4)
has been processed.
16. Mix gently but vortex and spin. Make aliquots of 100 μg for
fractionation and, optionally, take a separate 20 μg aliquot for
checking peptide labeling (see Note 5).
17. Dry in speed-vac and proceed to clean-up/fractionation or
store at À80
C.
3.6 Peptide BasicReversed-Phase
Fractionation
Peptide fractionation is necessary when handling complex samples
and allows to improve the number and the sequence coverage of
the identified proteins, but at the cost of increasing the number of
LC injections. Before fractionation, an optional step is to check by
LC-MS/MS the digestion and labeling (see Note 5).
1. Add 300 μL of 0.1% TFA to the dried labeled sample (~100 μg)
and vortex/sonicate for 5 min each at 25
C at 1200 rpm.
Repeat.
2. In the meantime, prepare the AcN/triethylamine solutions
with increasing amounts of AcN (300 μL of each solution per
column, see manufacturer brochure). Start with a wash solution of 5% AcN in trimethylamine and increase AcN sequentially by 2.5%, each up to 25%. Make a final elution solution of
50% AcN for very hydrophobic peptides for a total of nine
elution steps (see Note 6).
3. Remove the white cap and centrifuge the column for 2 min at
5000 Â g at 15
C.
4. Discard the liquid.
5. Remove the red cap and add 300 μL of AcN to the column,
close the cap and centrifuge for 2 min, 5000 Â g, 15
C.
6. Discard the liquid and repeat twice.
7. Add 300 μL of 0.1% TFA to the column.
Extracellular Vesicles from Mesenchymal Stem Cells
9
an average vortex once added to the samples. The leftover
reagent could be stored, tightly closed, at À20
C for future
experiments (see storage conditions in the manufacturer
brochure).
12. Incubate for 1 h at room temperature.
13. Just 5 min before the end of the incubation, prepare 5% HXAM
by adding 10 μL of 50% HXAM + 80 μL of H 2 O
(LC-MS) + 10 μL of 1 M TEAB. Vortex, spin, and then add
8 μL of 5% HXAM to the samples.
14. Incubate for 15 min at room temperature. Spin.
15. Transfer the entire contents of each labeling to the same 2-mL
vial. The final total volume should be ~1.7 mL containing a
total of 1000 μg of labeled peptides if all the channels have
been used and 100 μg of total protein per channel (see Note 4)
has been processed.
16. Mix gently but vortex and spin. Make aliquots of 100 μg for
fractionation and, optionally, take a separate 20 μg aliquot for
checking peptide labeling (see Note 5).
17. Dry in speed-vac and proceed to clean-up/fractionation or
store at À80
C.
3.6 Peptide BasicReversed-Phase
Fractionation
Peptide fractionation is necessary when handling complex samples
and allows to improve the number and the sequence coverage of
the identified proteins, but at the cost of increasing the number of
LC injections. Before fractionation, an optional step is to check by
LC-MS/MS the digestion and labeling (see Note 5).
1. Add 300 μL of 0.1% TFA to the dried labeled sample (~100 μg)
and vortex/sonicate for 5 min each at 25
C at 1200 rpm.
Repeat.
2. In the meantime, prepare the AcN/triethylamine solutions
with increasing amounts of AcN (300 μL of each solution per
column, see manufacturer brochure). Start with a wash solution of 5% AcN in trimethylamine and increase AcN sequentially by 2.5%, each up to 25%. Make a final elution solution of
50% AcN for very hydrophobic peptides for a total of nine
elution steps (see Note 6).
3. Remove the white cap and centrifuge the column for 2 min at
5000 Â g at 15
C.
4. Discard the liquid.
5. Remove the red cap and add 300 μL of AcN to the column,
close the cap and centrifuge for 2 min, 5000 Â g, 15
C.
6. Discard the liquid and repeat twice.
7. Add 300 μL of 0.1% TFA to the column.
Extracellular Vesicles from Mesenchymal Stem Cells
9