To begin, prepare proteome and phosphoproteome samples of the extracellular vesicles isolated from the urine samples using the EVtrap approach. Inject the samples into the trapped ion-mobility time-of-flight mass spectrometer through the liquid chromatography system. Separate the peptides into a 15-centimeter C18 column.
For proteomics analysis, acquire data using the dia-PASEF method with a mass range per ramp to span from 300 to 1200 mass-to-charge ratio and ion-mobility constant from 0.6 to 1.50 1/Knot. For phosphoproteomics analysis, acquire data using a dia-PASEF acquisition method. Set the mass range per ramp to span from 400 to 1550 mass-to-charge ratio in ion-mobility constant from 0.6 to 1.50 1/Knot.
Load the raw files into proteomic software. Set up the search parameters in the homo sapiens database. For Digest Type, select Specific, and under Enzymes, select TrypsinP.
Define the Peptide Length to a minimum of 7 and a maximum of 52 and allow two missed cleavages. Then set the Maximum Variable Modifications to 5. The Fixed Modification should be Carbamidomethyl at Cystine, while the Variable Modifications should include Acetyl Protein N-Terminal, Oxidation at Methionine, and Phosphorylation at Serine, Threonine, and Tyrosine.
Finally, set the false discovery rate for peptide spectrum match, peptide, and protein group to 0.01. In the LCMS and MS proteomic profiling, 2%of each sample revealed over 11, 000 unique peptides from approximately 2, 200 proteins. Notably, 72%of unique proteins were consistently found in all three replicates and 90 top EV markers and proteins were identified compared to the ExoCarta database.
Quantitative precision was confirmed with a low-medium coefficient of variation of 5.7%signifying high reproducibility and reliability. For phosphoproteomics, 98%of each peptide sample yielded 800 unique phosphopeptides and 350 phosphoproteins. The enrichment resulted in 72%phosphoserine, 22%phosphothreonine, and 6%phosphotyrosine peptides.
42%of phosphopeptides were identified in all replicates in a medium coefficient of variation of 21.8%was observed, indicating acceptable quantitative reproducibility.
