4. The recommendation is a single freeze–thaw cycle and storage
up to 1 year (À80
C) since these conditions have a low impact
on the concentration and size of vesicles.
5. KBr is used to solubilize lipoproteins and remove them from
the plasma.
6. The use of pools is not ideal but can be the only choice due
when there is sample limitation. In that case, validation should
be in individual biological samples.
7. IPG strips pH 4–7 are preferred for use because the majority of
EV proteins are distributed in this pH range.
8. If other equipment is used (e.g., IPGPhor 3), the electrophoresis protocol must be adapted.
9. Searches are restricted to human taxonomy allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as potential variable modification. Both the precursor
mass tolerance and the MS/MS tolerance were set at 0.3 and
0.4 Da, respectively, allowing one missed tryptic cleavage site.
10. With increasing temperature, urea decomposes to ammonia
and isocyanic acid. For this reason, it is necessary to temper
samples before diluting them with UA buffer.
11. Only less than 0.1% SDS (final concentration) is compatible
with the filter. For this reason, it is necessary to dilute the
sample many times with the UA buffer in order to reduce the
concentration.
12. The trypsin activity is affected by the concentration of urea.
Therefore, mix gently the washes with the pipette in order to
avoid possible remains of urea.
13. The HPLC system mentioned in this example is composed of
an Agilent 1200 capillary nano pump, a binary pump, a thermostated microinjector, and a micro switch valve.
14. The LTQ XL Orbitrap is operated in the positive ion mode
with a spray voltage of 1.8 kV. The spectrometric analysis is
performed in a data-dependent mode, acquiring a full scan
followed by ten MS/MS scans of the ten most intense signals
detected in the MS scan from the global list. The full MS (range
400–1650) is acquired in the Orbitrap with a resolution of
60,000. The MS/MS spectra are done in the linear ion trap.
Acknowledgments
The authors acknowledge support from the Spanish Ministry of
Science and Innovation [grants No. SAF2016-79662-R,
and PID2019-108727RB-I00], co-funded by the European
Regional Development Fund (ERDF). Financial support from the
22
Maria N. Barrachina and A ´ ngel Garcı ´a
up to 1 year (À80
C) since these conditions have a low impact
on the concentration and size of vesicles.
5. KBr is used to solubilize lipoproteins and remove them from
the plasma.
6. The use of pools is not ideal but can be the only choice due
when there is sample limitation. In that case, validation should
be in individual biological samples.
7. IPG strips pH 4–7 are preferred for use because the majority of
EV proteins are distributed in this pH range.
8. If other equipment is used (e.g., IPGPhor 3), the electrophoresis protocol must be adapted.
9. Searches are restricted to human taxonomy allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as potential variable modification. Both the precursor
mass tolerance and the MS/MS tolerance were set at 0.3 and
0.4 Da, respectively, allowing one missed tryptic cleavage site.
10. With increasing temperature, urea decomposes to ammonia
and isocyanic acid. For this reason, it is necessary to temper
samples before diluting them with UA buffer.
11. Only less than 0.1% SDS (final concentration) is compatible
with the filter. For this reason, it is necessary to dilute the
sample many times with the UA buffer in order to reduce the
concentration.
12. The trypsin activity is affected by the concentration of urea.
Therefore, mix gently the washes with the pipette in order to
avoid possible remains of urea.
13. The HPLC system mentioned in this example is composed of
an Agilent 1200 capillary nano pump, a binary pump, a thermostated microinjector, and a micro switch valve.
14. The LTQ XL Orbitrap is operated in the positive ion mode
with a spray voltage of 1.8 kV. The spectrometric analysis is
performed in a data-dependent mode, acquiring a full scan
followed by ten MS/MS scans of the ten most intense signals
detected in the MS scan from the global list. The full MS (range
400–1650) is acquired in the Orbitrap with a resolution of
60,000. The MS/MS spectra are done in the linear ion trap.
Acknowledgments
The authors acknowledge support from the Spanish Ministry of
Science and Innovation [grants No. SAF2016-79662-R,
and PID2019-108727RB-I00], co-funded by the European
Regional Development Fund (ERDF). Financial support from the
22
Maria N. Barrachina and A ´ ngel Garcı ´a