on their intensity (threshold: 2 Â 10
4 ), with dynamic exclusion
of 20 s, followed by their isolation with a window width of
Æ2 units of m/z, in a maximum time of 200 ms for fragmentation by HCD (high collision dissociation) with a normalized
collision energy (NCE) of 32%. MS/MS spectra were acquired
in positive mode.
8. Raw data were processed using Proteome Discoverer 2.4 software. Search was done against the last Uniprot protein databases (human and contaminants, Fig. 2b–d).
4 Notes
1. Acetone precipitation could be prolonged for up to 16 h.
2. If the sample does not completely dissolve, repeat this last step
by adding a small amount of TEAB (no more than 20 μL).
3. We strongly recommend checking the protein integrity by
SDS-PAGE and silver staining after BCA quantitation. Samples
presenting protein degradation should not be included in the
TMT study.
4. Scale down the amount of trypsin if necessary. Minimal recommended amount of total protein per channel is 20 μg. In any
case, always process the same amount of total protein per
channel.
5. LC analysis of peptide fractions is expensive and timeconsuming. Prior to fractionation, we recommend taking a
separate 20 μg aliquot and, after C-18 desalting, checking
labeling and peptide complexity by LC-MS/MS using a short
chromatographic gradient.
6. The number of elution steps can be scaled down depending on
the complexity of the sample (observed by SDS-PAGE). Never
leave the columns dry and keep wash eluates and sample flowthrough as a precaution in case there is a problem with the
binding of the peptides to the C18 resin.
7. We recommend dividing each fraction in three equal parts
(~100 μL each). One aliquot could be used for peptide quantitation (using, for instance, the peptide colorimetric quantitation assay); use the other two aliquots for LC-MS/MS analysis
of a known amount of peptides to avoid column overloading.
Acknowledgments
We thank M. Luisa Herna ´ez and M. Dolores Gutie ´rrez (from the
Proteomics Facility, Complutense University and Scientific Park
Foundation of Madrid, Spain) for their excellent MS data
acquisition.
Extracellular Vesicles from Mesenchymal Stem Cells
11
4 ), with dynamic exclusion
of 20 s, followed by their isolation with a window width of
Æ2 units of m/z, in a maximum time of 200 ms for fragmentation by HCD (high collision dissociation) with a normalized
collision energy (NCE) of 32%. MS/MS spectra were acquired
in positive mode.
8. Raw data were processed using Proteome Discoverer 2.4 software. Search was done against the last Uniprot protein databases (human and contaminants, Fig. 2b–d).
4 Notes
1. Acetone precipitation could be prolonged for up to 16 h.
2. If the sample does not completely dissolve, repeat this last step
by adding a small amount of TEAB (no more than 20 μL).
3. We strongly recommend checking the protein integrity by
SDS-PAGE and silver staining after BCA quantitation. Samples
presenting protein degradation should not be included in the
TMT study.
4. Scale down the amount of trypsin if necessary. Minimal recommended amount of total protein per channel is 20 μg. In any
case, always process the same amount of total protein per
channel.
5. LC analysis of peptide fractions is expensive and timeconsuming. Prior to fractionation, we recommend taking a
separate 20 μg aliquot and, after C-18 desalting, checking
labeling and peptide complexity by LC-MS/MS using a short
chromatographic gradient.
6. The number of elution steps can be scaled down depending on
the complexity of the sample (observed by SDS-PAGE). Never
leave the columns dry and keep wash eluates and sample flowthrough as a precaution in case there is a problem with the
binding of the peptides to the C18 resin.
7. We recommend dividing each fraction in three equal parts
(~100 μL each). One aliquot could be used for peptide quantitation (using, for instance, the peptide colorimetric quantitation assay); use the other two aliquots for LC-MS/MS analysis
of a known amount of peptides to avoid column overloading.
Acknowledgments
We thank M. Luisa Herna ´ez and M. Dolores Gutie ´rrez (from the
Proteomics Facility, Complutense University and Scientific Park
Foundation of Madrid, Spain) for their excellent MS data
acquisition.
Extracellular Vesicles from Mesenchymal Stem Cells
11