Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification

Abstract

Invasive nature and pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids have recently gained significant traction in the liquid biopsy field. EVs offer an essential "snapshot" of their precursor cells in real time and contain information-rich collection of nucleic acids, proteins, lipids, etc.The analysis of protein phosphorylation, as a direct marker of cellular signaling and disease progression, could be an important stepstone to successful liquid biopsy applications. Here, we introduce a rapid EV isolation method based on chemical affinity called EVtrap (Extracellular Vesicles Total Recovery and Purification) for EV phosphoproteomics analysis of human plasma. Incorporating EVtrap with high performance mass spectrometry (MS), we were able to identify over 16,000 unique peptides representing 2,238 unique EV proteins from just 5 μL plasma sample, including most known EV markers, with substantially higher recovery levels compared to ultracentrifugation. Most importantly, more than 5,500 unique phosphopeptides representing almost 1,600 phosphoproteins in EVs were identified using only 1 mL of plasma. Finally, we carried out quantitative EV phosphoproteomics analysis of plasma samples from patients diagnosed with chronic kidney disease or kidney cancer, identifying dozens of phosphoproteins capable of distinguishing disease states from healthy controls. The study demonstrates the potential feasibility of our robust analytical pipeline for cancer signaling monitoring by tracking plasma EV phosphorylation.

Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells

“…EVs enriched by EVtrap met required EV physical characteristics according to the MISEV 2018 criteria [ 2 ]. Size distribution measured was lower than 200 nm, underlying that EVtrap beads capture a small EV subset, as demonstrated in previous studies [ 25 , 26 ]. EVs were also shown by western blot to contain common well-characterized markers-PDCD6IP and TSG101 [ 28 ].…”

“…EVs from primary human brain microvascular endothelial cells were isolated using an EVtrap isolation method based on a chemical affinity capture approach, permitting to isolate small extracellular vesicles [ 25 , 26 ].…”

Proteomic Analysis of Exosomes for Discovery of Protein Biomarkers for Prostate and Bladder Cancer

“…Most immunocapture assays use monoclonal antibodies immobilized on a solid-phase (e.g., magnetic beads) to capture EVs that expose a specific ligand [ 57 , 58 , 59 , 60 ]. Other affinity isolation methods use chemical affinity [ 61 , 62 ] or annexin A5 which binds to phosphatidylserine moieties on the surface of most EVs [ 63 ]. Based on specific ligands or proteins, immunocapture can isolate subpopulations of EVs [ 21 , 64 ].…”

“…With ExoQuick or UC, ~300 [ 92 ] and ~100 proteins [ 86 ] were identified with ~32% and ~70% annotated as exosomal proteins, respectively. The highest number of exosomal proteins in plasma/serum reported so far was 2238 using immunoaffinity isolation with only 5 µL of plasma [ 61 ]; however, the GO term analysis showed that only 18.4% were annotated as exosomal proteins, suggesting issues with exosomal purity.…”

Extracellular Vesicles: New Endogenous Shuttles for miRNAs in Cancer Diagnosis and Therapy?

“…Careful consideration of the EVs isolation method must be taken into account when interpreting study results. The most common methods are a series of differential centrifugation steps, ultrafiltration, chemical affinity purification, and Exoquick [39][40][41] (Table 1). As demonstrated in conditional medium from cell culture the purification methods of EVs may heavily influence the yield, purity, and integrity of RNA extracted from EVs [42,43].…”