EV Sample Preparation for iTRAQ Proteomic Analysis
iTRAQ (isobaric Tags for Relative and Absolute Quantitation) proteomics is a quantitative mass spec approach in which up to eight protein samples are digested, their peptides labeled with isobaric tags, pooled, and analyzed together in a single multiplexed liquid chromatography–tandem mass spectrometry (LC-MS/MS) run. This reduces run-to-run variability, enables simultaneous comparison of protein abundance across multiple samples, and identifies cargo proteins that are enriched or depleted under different biological conditions.
This protocol describes how to prepare EV protein lysates for iTRAQ analysis.
Cushion-Density Gradient Ultracentrifugation Purification of Small and Large EVs
C-DGUC is the standard-practice protocol used in the Weaver lab for isolating high-purity small EVs from serum-free conditioned media. It captures the large EV fraction via ultracentrifugation, then gently collects small EVs in the clarified supernatant by ultracentrifugation onto an iodixanol cushion. This small EV fraction is further refined by density gradient ultracentrifugation, which separates distinct small EV subpopulations and removes any remaining non-vesicular contaminants. This method avoids potential sEV damage and aggregation caused by repeated pelleting and achieves high-purity EV isolation, enabling their use in downstream functional assays.
Researchers should independently evaluate whether EV purity or EV quantity is more important for their downstream applications and select an EV isolation method accordingly.
Small EV Purification by Standard Ultracentrifugation +/- Density Gradient
This is a traditional method for isolating large and small EVs from conditioned media. It uses a series of differential-speed ultracentrifugation (DUC) steps to isolate cell debris, large EVs, and small EVs from the conditioned media, yielding a crude small EV preparation. If a more refined isolation of small EVs is desired, the protocol continues by running the crude EV fraction through a discontinuous iodixanol density gradient ultracentrifugation (DGUC). This DUC-DGUC method is a widely used and effective approach for small EV isolation, and the DUC portion of the protocol is a good way to get started before trying density gradient methods. However, it is important to note that the many pelleting steps can cause vesicle aggregation, loss of integrity, and reduced yield due to incomplete resuspension.
For the gold standard EV isolation method used in the Weaver lab, refer to the Cushion-Density Gradient Ultracentrifugation protocol.
SYPRO Ruby Flourescent Gel Staining of Protein Concentration
This protocol provides an alternative method for quantifying protein in EVs. Compared with standard colorimetric assays such as BCA assay or microBCA, which may overestimate EV protein content and can be influenced by lipids or other sample components, this method determines protein concentration by fluorescent staining of the full lane following SDS-PAGE, using the known quantities of the protein bands in the BenchMark ladder as reference standards.
In our experience, this provides a more accurate estimate of EV protein input for downstream applications such as Western blotting and mass spectrometry–based proteomics.