Recently, the team of Lifang Yang at the Cancer Research Center of Eastern Virginia Medical School in the United States proposed an optimized differential ultracentrifugation method that combines THP polymer reduction with alkaline washing, so as to improve the isolation of EVs and remove THP and other common protein contaminants.
When applied to expressed prostatic secretions in urine, the method achieved relative enrichment of EV proteins known to be associated with prostate cancer. It provides a highly promising strategy for the proteomic analysis of urinary extracellular vesicles.
Background
The isolation of extracellular vesicles (EVs) from patient samples, followed by molecular analysis, is a widely used strategy for understanding vesicle biology and advancing biomarker development. Expressed prostatic secretions in urine are a tumour-proximal fluid that has attracted considerable attention in liquid biopsy as a source of potential prostate cancer (PCa) biomarkers. Proteomic analysis of urinary extracellular vesicles (uEVs) from the expressed prostatic secretions (EPS-urine) of PCa patients holds promise for developing non-invasive PCa biomarkers that improve the clinical management of the disease. Isolating EVs from urine, however, presents substantial inherent challenges: standard EV isolation methods such as differential ultracentrifugation (dUC) give low recovery, and the protein contaminants co-isolated with EVs reduce the detection depth for low-abundance uEV proteins in typical mass spectrometry (MS) analyses. The research team therefore developed an improved dUC method that uses alkaline washing to raise recovery and reduce protein contaminants, applied it to the analysis of urine samples from prostate cancer patients, and explored its application in prostate cancer-associated urinary EV proteomics.
Results
Previous studies have shown that reducing agents can effectively depolymerize the THP (Tamm-Horsfall protein) matrix in low-speed pellets so as to release THP-coated EVs; THP oligomers/monomers and other abundant proteins, however, can co-pellet with EVs during the subsequent high-speed ultracentrifugation (UC). Bypassing the THP depolymerization step avoids these contaminants, but at the cost of losing part of the urinary EVs. Sodium carbonate washing can be used to separate integral membrane proteins from peripheral membrane proteins. The researchers therefore reasoned that introducing an alkaline sodium carbonate wash could improve EV purity by altering the protonation state of the ionizable groups on EVs and their bound proteins, thereby reducing non-covalent interactions. The effectiveness of the alkaline wash step was evaluated by comparing four uEV isolation methods: (1) DTT treatment of the low-speed pellet (20,000 g) and alkaline washing of the high-speed pellet (175,000 g); (2) DTT treatment of the low-speed pellet and PBS washing of the high-speed pellet; (3) bypassing DTT treatment of the low-speed pellet and applying alkaline washing to the high-speed pellet; and (4) bypassing DTT treatment of the low-speed pellet and washing the high-speed pellet with PBS. These four methods are referred to as DTT-ALK, DTT-PBS, ALK and PBS respectively.
Schematic diagram of the dUC method
Comparison of multiple optimized differential ultracentrifugation strategies for isolating uEVs
Compared with the other methods, THP contamination in uEVs prepared by DTT-ALK was significantly reduced, raising the CD9/THP ratio by 37–42 fold and the TSG101/THP ratio by 29–92 fold. The uEVs obtained by all methods contained characteristic round vesicular structures. Importantly, the uEVs isolated under alkaline conditions (DTT-ALK and ALK) showed intact membrane-enclosed vesicles, indicating that alkaline washing does not disturb the structural integrity of uEVs.
Characterization of dUC-enriched EPS-urine extracellular vesicles
All methods enriched small particles with a size distribution between 50 and 150 nm, a size range consistent with that expected for small EVs; there was no significant difference in mean and mode particle size between the particles isolated by the DTT-ALK and PBS methods, while the mean and mode particle sizes for DTT-ALK and PBS were significantly lower than for DTT-PBS. Overall mean particle content was lowest with the DTT-PBS method, and the mean protein/particle ratio for DTT-PBS (2.46 ± 0.80 SD) was significantly higher than for the other methods (DTT-ALK 1.16 ± 0.14 SD; PBS 1.00 ± 0.15 SD), indicating that the uEVs isolated by this method had the lowest purity. Taken together, these findings indicate that the DTT-ALK method offers the best uEV isolation efficiency and structural integrity, with reduced contamination by urinary THP.
Nanoparticle tracking analysis of EVs isolated from EPS-urine
Proteomic Analysis of uEVs
To reveal the protein composition of uEVs, the researchers performed in-depth proteomic analysis of uEVs isolated by the DTT-ALK, DTT-PBS and PBS methods. Label-free LC-MS/MS quantification was carried out on the uEV P4 fraction. The average number of proteins identified was highest in uEVs prepared by the DTT-ALK method (1,786 ± 9.8 SD), followed by PBS (1,598 ± 19.7 SD) and DTT-PBS (1,275 ± 2.9 SD). Principal component analysis showed that the EVs isolated by DTT-ALK more closely resembled those from PBS than those from DTT-PBS. These results highlight the differing protein content produced by each uEV isolation method and indicate that uEVs isolated by DTT-ALK and PBS show relatively greater similarity.
Comparison of the proteomes identified by LC-MS/MS across different EV isolation methods
Relative Enrichment of Prostate Cancer-Associated and Tissue-Specific Proteins
The researchers compared the relative enrichment of prostate-specific and prostate cancer-associated biomarkers across the different methods, and compiled a list of 27 PCa biomarker proteins that were present in the study dataset and had been confirmed in the literature to occur in uEVs. In addition, the study dataset was searched against the TiGER (Tissue-specific Gene Expression and Regulation) database for transcriptomes of genes preferentially expressed in the prostate; the resulting list contained 33 putative PCa biomarkers.
The results showed that the average intensity of 27 of the 33 proteins differed significantly among the three methods. Secreted proteins including prostatic acid phosphatase (ACPP/PAP), kallikrein 2 (KLK2) and prostate-specific antigen (KLK3/PSA) were reduced in uEVs isolated by DTT-ALK and enriched in the DTT-PBS method. In contrast, the DTT-ALK method significantly enriched intracellular protein biomarkers including the metalloreductases STEAP2/4, anoctamin 7 (ANO7), glutamate carboxypeptidase 2 (FOLH1/PSMA), retinol dehydrogenase 11 (RDH11) and dipeptidyl peptidase 4 (DPP4), as well as intracellular proteins such as peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5), fatty acid synthase (FASN), sorbitol dehydrogenase (SORD), protein NDRG1 (NDRG1) and copine-4 (CPNE4). These results once again indicate that the DTT-ALK method can enrich uEVs derived from the prostate.
Ingenuity Pathway Analysis (IPA) was used to identify PCa-related signalling pathways in the uEVs isolated by all three methods; the results showed that uEVs isolated using the DTT-ALK method were enriched for prostate-derived and PCa-associated proteins.
Conclusions
This study developed an effective EV isolation method that integrates DTT treatment and alkaline washing into dUC in order to isolate uEVs of higher purity and higher yield. The method represents an easy-to-use and efficient dUC strategy that depletes abundant urinary proteins as well as the proteins bound to the uEV protein corona through non-covalent interactions, and thereby extracts the uEVs. This preparation enables comprehensive, deep proteomic analysis of prostate-derived uEVs by LC-MS/MS. In addition, DTT treatment and alkaline washing can be incorporated into other uEV isolation workflows to further improve uEV purity. This approach may improve the subsequent discovery and development of biomarkers in clinical urine samples.
Reference
Correll V L, Otto J J, Risi C M, et al. Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in‐depth proteomic analysis[J]. Journal of extracellular vesicles, 2022, 11(2): e12184.
