To begin, take the dried extracellular vesicle sample obtained from the human urine using the EV trap approach. Prepare a fresh lysis buffer with the shown components. Then add 100 microliters of lysis buffer to solubilize the dried EV sample.
Heat the sample at 95 degrees Celsius for 10 minutes while shaking at 1, 100 RPM. After cooling the sample to room temperature, dilute it fivefold by adding 400 microliters of 50 millimolar TEAB. Measure the protein concentration using a BCA assay kit according to the manufacturer's instructions.
Add trypsin and lysine C mix to the sample an incubate at 37 degrees celsius overnight with shaking at 1, 100 RPM. Then add 50 microliters of 10%trifluoroacetic acid or TFA to acidify the sample. Further, add 600 microliters of ethyl acetate to the samples and vortex the mixture for two minutes.
Centrifuge the sample for three minutes at 20, 000 G and carefully remove the upper layer without disturbing the interface. Dry the aqueous phase using a vacuum centrifuge concentrator. Now resuspend the dried sample in 200 microliters of 0.1%TFA to acidify peptides.
To desalt the sample, use a CA18 desalting tip and condition it with 200 microliters of 0.1%TFA and 80%acetonitrile, followed by two washes with 200 microliters of 0.1%TFA. Load the acidified peptide sample into the tip and wash it three times with 200 microliters of 0.1%TFA. Elute the peptides with 200 microliters of 0.1%TFA and 80%acetonitrile.
After drawing the eluent as demonstrated, resuspend the dried to phosphoproteomics sample in 200 microliters of loading buffer for phosphopeptide enrichment. Add 50 microliters of the beads to the sample and vigorously shake for 20 minutes at room temperature. Load the sample with the beads into the fritted tip and centrifuge for one minute at 100 G.Wash the tip successively with 200 microliters of loading buffer, washing buffers one and two.
Place the tip with beads into a new tube to collect the eluded phosphopeptides. Add 50 microliters of elution buffer to the tip and centrifuge for two minutes at 20 G.Then dry the eluded phosphopeptides using a vacuum centrifuge concentrator.
