Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles secreted by all types of cells and are present in biofluids such as blood, urine, saliva, etc.^1,2,3,4. EVs carry a cargo of diverse bioactive molecules which reflect the physiological and pathological state of their host cells and, therefore function as crucial factors in disease progression^4,5,6. Moreover, extensive studies have established that EV-based disease markers can be ide....

All urine samples were collected from healthy individuals after informed consent. The experiments were compliant with all ethical standards involving human samples and conform to the guidelines from Purdue University Human Research Protection Program.

1. Sample collection

1. Centrifuge 12 mL of urine sample in a 15 mL conical centrifuge tube for 10 min at 2,500 x g, 4 °C to remove cell debris and large apoptotic bodies.
2. Transfer 10 mL of the su.......

This protocol demonstrates a comprehensive workflow from the isolation of EVs to downstream proteomics and phosphoproteomics analyses (Figure 1). The triplicate urine samples were subjected to EV isolation. The isolated EVs were characterized by western blotting and subsequently processed for mass spectrometry-based proteomics sample preparation including protein extraction, enzymatic digestion, and peptide cleanup. For phosphoproteomics analysis, the phosphopeptides were further enriched ba.......

Effective EV isolation is an essential prerequisite to detecting low-abundant proteins and phosphoproteins in EVs. Despite the development of numerous methods to fulfill this need, the majority still suffer from limitations such as poor recovery or low reproducibility, which impede their utilization in large-scale studies and routine clinical settings. DUC is generally considered as the most common method for EV isolation, and the additional washing steps are normally applied to help increase the purity of target EVs

This work has been funded in part by NIH grants 3RF1AG064250 and R44CA239845.