Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation.

Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease, and healthy control individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.

Hands-Free Proteomic Profiling of Urinary Extracellular Vesicles with a High-Throughput Automated Workflow.

Extracellular vesicles (EVs) have emerged as a promising source of disease biomarkers for noninvasive early stage diagnoses, but a bottleneck in EV sample processing restricts their immense potential in clinical applications. Existing methods are limited by a low EV yield and integrity, slow processing speeds, low sample capacity, and poor recovery efficiency. We aimed to address these issues with a high-throughput automated workflow for EV isolation, EV lysis, protein extraction, and protein denaturation. The automation can process clinical urine samples in parallel, resulting in protein-covered beads ready for various analytical methods, including immunoassays, protein quantitation assays, and mass spectrometry. Compared to the standard manual lysis method for contamination levels, efficiency, and consistency of EV isolation, the automated protocol shows reproducible and robust proteomic quantitation with less than a 10% median coefficient of variation. When we applied the method to clinical samples, we identified a total 3,793 unique proteins and 40,380 unique peptides, with 992 significantly upregulated proteins in kidney cancer patients versus healthy controls. These upregulated proteins were found to be involved in several important kidney cancer metabolic pathways also identified with a manual control. This hands-free workflow represents a practical EV extraction and profiling approach that can benefit both clinical and research applications, streamlining biomarker discovery, tumor monitoring, and early cancer diagnoses.

Quantitative proteomics and phosphoproteomics of urinary extracellular vesicles define putative diagnostic biosignatures for Parkinson's disease.

BACKGROUND: Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been recognized as genetic risk factors for Parkinson's disease (PD). However, compared to cancer, fewer genetic mutations contribute to the cause of PD, propelling the search for protein biomarkers for early detection of the disease. METHODS: Utilizing 138 urine samples from four groups, healthy individuals (control), healthy individuals with G2019S mutation in the LRRK2 gene (non-manifesting carrier/NMC), PD individuals without G2019S mutation (idiopathic PD/iPD), and PD individuals with G2019S mutation (LRRK2 PD), we applied a proteomics strategy to determine potential diagnostic biomarkers for PD from urinary extracellular vesicles (EVs). RESULTS: After efficient isolation of urinary EVs through chemical affinity followed by mass spectrometric analyses of EV peptides and enriched phosphopeptides, we identify and quantify 4476 unique proteins and 2680 unique phosphoproteins. We detect multiple proteins and phosphoproteins elevated in PD EVs that are known to be involved in important PD pathways, in particular the autophagy pathway, as well as neuronal cell death, neuroinflammation, and formation of amyloid fibrils. We establish a panel of proteins and phosphoproteins as novel candidates for disease biomarkers and substantiate the biomarkers using machine learning, ROC, clinical correlation, and in-depth network analysis. Several putative disease biomarkers are further partially validated in patients with PD using parallel reaction monitoring (PRM) and immunoassay for targeted quantitation. CONCLUSIONS: These findings demonstrate a general strategy of utilizing biofluid EV proteome/phosphoproteome as an outstanding and non-invasive source for a wide range of disease exploration.

Parkinson's disease (PD) is a progressive neurological disorder that affects body movement because some brain cells stop producing the chemical dopamine. PD is often not diagnosed until it has advanced, making early detection crucial. To enable early detection, we investigated tiny packages called extracellular vesicles released from a variety of cells, including the brain cells, that can be found in urine as a potential source for diagnosing PD. These tiny packages contain different kinds of molecules from inside the cells. We analyzed urine samples from 138 individuals and found several proteins involved in PD development that could be biological indicators for early detection of the disease. We used various techniques to make sure that our findings were accurate. Our study suggests that looking at these proteins in urine could be a good way to detect PD in a non-invasive manner.