One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles

Liu, Yi-Kai; Wu, Xiaofeng; Hadisurya, Marco; Li, Li; Kaimakliotis, Hristos; Iliuk, Anton; Tao, W. Andy

doi:10.1021/acs.jproteome.3c00361

Cited by 3 publications

(1 citation statement)

References 72 publications

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“…These magnetic beads can isolate the EVs with minimal sample losses with the trade-off of a lower capacity. They have also been successfully utilized into manual workflows that allow the entire EV analytical pipeline to be performed within a single tube . Unfortunately, the greater portion of work required to be performed by hand leads to more difficult processing, although the high efficiency benefits of this method often justify the drawbacks.…”

Section: Introductionmentioning

confidence: 99%

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

Lee,

Hadisurya,

Luo

et al. 2023

J. Am. Soc. Mass Spectrom.

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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 handsfree 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.

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Section: Introductionmentioning

confidence: 99%

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

Lee,

Hadisurya,

Luo

et al. 2023

J. Am. Soc. Mass Spectrom.

Self Cite

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One-Pot Sequential Enrichment of Urinary Extracellular Vesicle and miRNAs Identifies a Noninvasive Biomarker Panel for Prostate Cancer Diagnosis

Liu,

Zhang,

Wei

et al. 2024

Anal. Chem.

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Extracellular vesicles (EVs) offer promising noninvasive alternatives for convenient and noninvasive prostate cancer (PCa) diagnosis, but inefficient EV enrichment and cargo extraction hinder discovery and validation for their clinical applications. Here, we present an integrated pipeline based on functionalized magnetic beads to streamline and enhance the efficiency of urinary EV miRNA analysis. EVs are first enriched on amphiphilic magnetic beads through chemical affinity, followed by EV lysis and the isolation of miRNAs through solid phase extraction. The new pipeline demonstrated a more than 10-fold increase in urine EV miRNA extraction efficiency compared to the traditional ultracentrifugation combined TRIzol method while reducing the sample processing time to within 1 h. The one-bead strategy further allowed us to automate the procedure on a 96-channel instrument. We applied the pipeline to analyze urine samples from 108 benign prostatic hyperplasia (BPH) controls and 92 PCa cases. Among 195 miRNA biomarkers from the literature, we prioritized 18 miRNAs for quantification and successfully validated 12 miRNAs with a ratio-based normalized method. The quantification data from BPH controls and PCa cases in the training set were subjected to a machine learning analysis of Random Forest, through which we generated a five-miRNA panel consisting of miR-148a-5p, miR-21−5p, miR-181a-5p, miR-222−3p, and miR-100−5p. This panel showed high sensitivity (89%) and specificity (72%) in the test set, highlighting immense potential of this streamlined pipeline for noninvasive diagnosis.

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High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array

Feng,

Shen,

Zhang

et al. 2024

Nat Protoc

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One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles

Cited by 3 publications

References 72 publications

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

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

One-Pot Sequential Enrichment of Urinary Extracellular Vesicle and miRNAs Identifies a Noninvasive Biomarker Panel for Prostate Cancer Diagnosis

High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array

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