High Throughput Isolation and Data Independent Acquisition Mass Spectrometry (DIA-MS) of Urinary Extracellular Vesicles to Improve Prostate Cancer Diagnosis

Abstract

Proteomic profiling of extracellular vesicles (EVs) represents a promising approach for early detection and therapeutic monitoring of diseases such as cancer. The focus of this study was to apply robust EV isolation and subsequent data-independent acquisition mass spectrometry (DIA-MS) for urinary EV proteomics of prostate cancer and prostate inflammation patients. Urinary EVs were isolated by functionalized magnetic beads through chemical affinity on an automatic station, and EV proteins were analyzed by integrating three library-base analyses (Direct-DIA, GPF-DIA, and Fractionated DDA-base DIA) to improve the coverage and quantitation. We assessed the levels of urinary EV-associated proteins based on 40 samples consisting of 20 cases and 20 controls, where 18 EV proteins were identified to be differentiated in prostate cancer outcome, of which three (i.e., SERPINA3, LRG1, and SCGB3A1) were shown to be consistently upregulated. We also observed 6 out of the 18 (33%) EV proteins that had been developed as drug targets, while some of them showed protein-protein interactions. Moreover, the potential mechanistic pathways of 18 significantly different EV proteins were enriched in metabolic, immune, and inflammatory activities. These results showed consistency in an independent cohort with 20 participants. Using a random forest algorithm for classification assessment, including the identified EV proteins, we found that SERPINA3, LRG1, or SCGB3A1 add predictable value in addition to age, prostate size, body mass index (BMI), and prostate-specific antigen (PSA). In summary, the current study demonstrates a translational workflow to identify EV proteins as molecular markers to improve the clinical diagnosis of prostate cancer.

In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum

“…46 Zhang et al, performed proteomic analysis of urinary EVs from prostate cancer and prostate inflammation patients using a DIA-constructed spectral library and identified 18 EV proteins that were significantly differentially expressed. 47 Halal et al, also applied library-based DIA to profile circulating EVs from glioma patients and healthy controls, achieving a coverage of 4054 proteins. 48 Chutipongtanate and Greis also previously reported the application of a curated library-based DIA for urinary EVs, detecting 888 proteins.…”

“…Zheng et al, demonstrated the potential of DIA-MS to validate circulating extracellular vesicles biomarkers in colorectal cancer patient samples . Zhang et al, performed proteomic analysis of urinary EVs from prostate cancer and prostate inflammation patients using a DIA-constructed spectral library and identified 18 EV proteins that were significantly differentially expressed . Halal et al, also applied library-based DIA to profile circulating EVs from glioma patients and healthy controls, achieving a coverage of 4054 proteins .…”

Prostate cancer-derived small extracellular vesicle proteins: the hope in diagnosis, prognosis, and therapeutics

“…Gamma-glutamyltransferase 1 was shown to be expressed at higher levels in urinary sEVs and tissues of PCa patients [ 145 ]. Zhang reported that urine sEV protein markers (SERPINA3, LRG1, and SCGB3A1) were higher in PCa patients (n = 20) than in healthy controls (n = 20), with SERPINA3 showing the highest correlation in distinguishing patients from healthy individuals [ 147 ]. Additionally, FABP5 was found to be not only higher in the PCa group compared with the control group, but also significantly correlated with Gleason grading in urinary sEVs [ 146 ].…”

The Potential of Extracellular Matrix- and Integrin Adhesion Complex-Related Molecules for Prostate Cancer Biomarker Discovery

“…In prostate cancer, the exosomes from different compartments were studied [145]. For example, the potential of urinary EVs [146] and plasma-derived exosomes [147] was explored. In a recent article, the authors did the proteomic analysis of EVs isolated from the blood sera of prostate cancer patients, healthy donors, patients with hypertrophic disease, and post-prostatectomy disease-free cases.…”