Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles

Nguyen, Ai; Wang, Tingting; Turko, Illarion V.

doi:10.1016/j.jprot.2021.104359

Cited by 3 publications

(6 citation statements)

References 16 publications

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“…Once identified, the selected peptides could be used for EV isolation. 32 Iaccino E. et al selected Id peptides by screening a phage display library to bait the Ig-BCR expressed by 5T33MM cells. By FACS, FITC-conjugated Id peptides detected MM-released exosomes in the serum of 5T33MMengrafted mice earlier than serum paraprotein, and the level of MM-released exosomes was correlated with tumor progression.…”

Section: ■ Resultsmentioning

confidence: 99%

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Phage Display Screening of Anchor Peptides for Red Blood Cell-Derived Extracellular Vesicles

Xu,

Xia

et al. 2024

ACS Omega

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Extracellular vesicles (EVs) are increasingly used for disease diagnosis and treatment. Among them, red blood cellderived EVs (RBC-EVs) have attracted great attention due to their abundant sources and low risks of gene transfer (RBC-EVs lack nuclear and mitochondrial DNA). Here, we first revealed the high expression level of membrane protein solute carrier family 4 member 1 (SLC4A1) in RBC-EVs through proteomic analysis. We then identified several binding peptides with high affinity for the SLC4A1 extracellular domain (SLC4A1-EC) from phage display library screening. A high affinity of SLC4A1-EC and the three peptides (XRB2, XRE4, and XRH7) were assessed in vitro using surface plasmon resonance analysis and SDS−polyacrylamide gel electrophoresis (SDS−PAGE). The binding sites of SLC4A1-EC and polypeptides were further predicted by LigPlot + analysis, and the results showed that these three polypeptides could bind to part of the hydrophobic residues of SLC4A1-EC. The binding efficiency of the anchor peptides to the RBC-EVs was further verified by flow cytometry and fluorescence imaging. In conclusion, we successfully screened three specific RBC-EV-targeting peptides which could potentially be utilized for isolating RBC-derived EVs from serum samples. More importantly, this peptide could be coupled with targeting peptides to modify RBC-EVs for drug delivery. Our work will provide a viable method for optimizing the function of RBC-EVs.

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Section: ■ Resultsmentioning

confidence: 99%

“…Nguyen A.et al used purified EVs as bait for screening a phage display peptide library. Once identified, the selected peptides could be used for EV isolation . Iaccino E. et al selected Id peptides by screening a phage display library to bait the Ig-BCR expressed by 5T33MM cells.…”

Section: Resultsmentioning

confidence: 99%

Phage Display Screening of Anchor Peptides for Red Blood Cell-Derived Extracellular Vesicles

Xu,

Xia

et al. 2024

ACS Omega

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“…Calibration curves were generated for each analyte in the panel to encompass the concentration range typically observed for normal human serum (calibrators 1–6) and at points two and four times beyond that, as may be observed with increased levels of circulating EVs in various disease states (calibrators 7 and 8) (Nguyen et al., 2021 ; Povero et al., 2020 ; Sehrawat et al., 2021 ). The required range varied considerably between positive and negative EV markers: Albumin was validated between 2.0 and 80 pmol/ml while this range was 100‐fold less for CD9 (Table 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…The concentration range over which the assay was validated comprised eight calibrators to cover concentrations observed in healthy donor serum and beyond to increased levels that may be observed in various disease contexts (Nguyen et al., 2021 ; Povero et al., 2020 ; Sehrawat et al., 2021 ). For all analytes, calibration curves were linear (all r 2 > 0.99) and the assay exhibited good precision and accuracy.…”

Section: Discussionmentioning

confidence: 99%

“…Few studies have previously employed targeted LC-MS/MS assays to assess purity of EVs from blood (Park et al, 2021;Wang et al, 2017), and have been useful in the development of novel isolation strategies (Nguyen et al, 2021) or to gain insight to membrane origin of circulating vesicles (Zhang et al, 2020). Of these studies, only that by Park et al (2021) was performed on clinically relevant volumes of sample (100 µl plasma, while others used up to 200 ml).…”

Section: Introductionmentioning

confidence: 99%

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Addressing MISEV guidance using targeted LC‐MS/MS: A method for the detection and quantification of extracellular vesicle‐enriched and contaminant protein markers from blood

Newman

Useckaite

Rowland

2022

J of Extracellular Bio

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Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and nonvesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations.

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Isolation protocols and mitochondrial content for plasma extracellular vesicles

Nguyen

Turko

2022

Anal Bioanal Chem

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Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles

Cited by 3 publications

References 16 publications

Phage Display Screening of Anchor Peptides for Red Blood Cell-Derived Extracellular Vesicles

Phage Display Screening of Anchor Peptides for Red Blood Cell-Derived Extracellular Vesicles

Addressing MISEV guidance using targeted LC‐MS/MS: A method for the detection and quantification of extracellular vesicle‐enriched and contaminant protein markers from blood

Isolation protocols and mitochondrial content for plasma extracellular vesicles

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