| thaw sample in slushed ice |
|
input
CSF volume: 400 μL (optimal). |
∼1 h for 400 μL CSF |
| 20K EV enrichment |
proteome characterization
of raw CSF and/or 20K EVs |
add PBS to 1000 μL final
volume |
∼1.5 h |
| centrifuge 20,000g for 30 min |
| wash with PBS (15 min) |
| transfer supernatant (until 20 μL) to a new tube; add PBS to 1000 μL and mix |
| centrifuge 20,000g for 30 min |
| discard supernatant–leave 20 μL incl pellet (10 min) |
| |
proceed to digestion if CSF biomarker
exploration is the main research aim |
| 120K EV enrichment |
proteomic
characterization of 120K EVs |
use supernatant collected
after the first 20k centrifugation |
∼4–5 h |
| balance
samples and place in rotor, 30 min |
| centrifuge 120,000g for 90 min |
| wash with PBS (15 min) as described
above |
| balance samples and place in rotor, 30 min |
| centrifuge 120,000g for 90 min |
| discard supernatant–leave 20 μL incl pellet (10 min) |
| tryptic digestion |
|
input volume: 20 μL pellet
from raw CSF, 20K and/or 120K EV fractionation. |
PAC-based on-bead protocol: 2 h |
| sample limitation or low protein input, chose urea-based in-solution |
urea-based in-solution: 1 h |
| if high protein input, chose PAC-based on-bead digest |
both |
| +1 h Lys-C incubation + overnight incubation
with trypsin |
