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Accessing the proteome of extracellular vesicles via rapid acoustic isolation of a minute human blood plasma sample

Havers, Megan LU orcid ; Scott, Aaron M. LU ; Ortenlöf, Niklas LU ; Welinder, Charlotte LU ; Ekström, Simon LU ; Baasch, Thierry LU ; Evander, Mikael ; Lenshof, Andreas LU orcid ; Gram, Magnus LU orcid and Laurell, Thomas LU (2025) In Analytica Chimica Acta 1379.
Abstract

Background: Despite substantial progress in the field of mass spectrometry, there remain barriers to measuring the extracellular vesicles (EVs) proteome in blood plasma. Recent work has shown that isolating EVs can make it possible to detect proteins that have low abundance in plasma. Commonly used EV isolation methods either require large sample volumes and long ultracentrifugation times, or else result in population bias via targeted isolation. There is a great need for fast and easy methods to isolate EVs from small volumes of plasma, <10 μL, enabling biomarker discovery, e.g. in biobanked samples, where mass spectrometry can play an important role. Results: We unveil the extracellular vesicle proteome by using seed particle... (More)

Background: Despite substantial progress in the field of mass spectrometry, there remain barriers to measuring the extracellular vesicles (EVs) proteome in blood plasma. Recent work has shown that isolating EVs can make it possible to detect proteins that have low abundance in plasma. Commonly used EV isolation methods either require large sample volumes and long ultracentrifugation times, or else result in population bias via targeted isolation. There is a great need for fast and easy methods to isolate EVs from small volumes of plasma, <10 μL, enabling biomarker discovery, e.g. in biobanked samples, where mass spectrometry can play an important role. Results: We unveil the extracellular vesicle proteome by using seed particle enhanced acoustic trapping to purify EVs from minute blood plasma samples (8 μL) in 6 min per sample. The differential mass spectrometry results find proteins which are significantly enriched (FDR-adjusted p-values<0.05) in acoustically trapped samples compared to raw (unprocessed) plasma, more than two thirds of those proteins have been associated with EVs previously. Additionally, we are able to increase the depth of analysis by detecting 51 low abundance proteins not detected in raw plasma, half of which are tagged with the gene ontology (GO) tag “extracellular exosome” (GO:0070062). Finally, we validate the novel use of neutrally charged silica seed particles paired with a washing flowrate of 200 μL/min, enabling the processing time to be halved and finding the same proteome as for tried-and-tested polystyrene seed particles with washing at 30 μL/min. Significance: Our microfluidics-based approach to EV isolation enables rapid processing of an individual minute blood plasma sample, demonstrating that personal proteomic information associated with EVs can be detected when acoustic trapping is used as a pre-processing step. By applying this technique to plasma from patient cohorts or mouse models, future studies may offer new insights into the role of EVs in the progression of diseases and reveal new diagnostic targets in the proteomic cargo of EVs.

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author
; ; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Acoustic trapping, Extracellular vesicles, Mass spectrometry, Microfluidics, Plasma, Proteomics
in
Analytica Chimica Acta
volume
1379
article number
344661
publisher
Elsevier
external identifiers
  • pmid:41167904
  • scopus:105017862799
ISSN
0003-2670
DOI
10.1016/j.aca.2025.344661
language
English
LU publication?
yes
id
90ed6f05-e1b3-4bb6-b180-11e32521378c
date added to LUP
2025-11-20 14:22:10
date last changed
2025-11-21 03:04:05
@article{90ed6f05-e1b3-4bb6-b180-11e32521378c,
  abstract     = {{<p>Background: Despite substantial progress in the field of mass spectrometry, there remain barriers to measuring the extracellular vesicles (EVs) proteome in blood plasma. Recent work has shown that isolating EVs can make it possible to detect proteins that have low abundance in plasma. Commonly used EV isolation methods either require large sample volumes and long ultracentrifugation times, or else result in population bias via targeted isolation. There is a great need for fast and easy methods to isolate EVs from small volumes of plasma, &lt;10 μL, enabling biomarker discovery, e.g. in biobanked samples, where mass spectrometry can play an important role. Results: We unveil the extracellular vesicle proteome by using seed particle enhanced acoustic trapping to purify EVs from minute blood plasma samples (8 μL) in 6 min per sample. The differential mass spectrometry results find proteins which are significantly enriched (FDR-adjusted p-values&lt;0.05) in acoustically trapped samples compared to raw (unprocessed) plasma, more than two thirds of those proteins have been associated with EVs previously. Additionally, we are able to increase the depth of analysis by detecting 51 low abundance proteins not detected in raw plasma, half of which are tagged with the gene ontology (GO) tag “extracellular exosome” (GO:0070062). Finally, we validate the novel use of neutrally charged silica seed particles paired with a washing flowrate of 200 μL/min, enabling the processing time to be halved and finding the same proteome as for tried-and-tested polystyrene seed particles with washing at 30 μL/min. Significance: Our microfluidics-based approach to EV isolation enables rapid processing of an individual minute blood plasma sample, demonstrating that personal proteomic information associated with EVs can be detected when acoustic trapping is used as a pre-processing step. By applying this technique to plasma from patient cohorts or mouse models, future studies may offer new insights into the role of EVs in the progression of diseases and reveal new diagnostic targets in the proteomic cargo of EVs.</p>}},
  author       = {{Havers, Megan and Scott, Aaron M. and Ortenlöf, Niklas and Welinder, Charlotte and Ekström, Simon and Baasch, Thierry and Evander, Mikael and Lenshof, Andreas and Gram, Magnus and Laurell, Thomas}},
  issn         = {{0003-2670}},
  keywords     = {{Acoustic trapping; Extracellular vesicles; Mass spectrometry; Microfluidics; Plasma; Proteomics}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Analytica Chimica Acta}},
  title        = {{Accessing the proteome of extracellular vesicles via rapid acoustic isolation of a minute human blood plasma sample}},
  url          = {{http://dx.doi.org/10.1016/j.aca.2025.344661}},
  doi          = {{10.1016/j.aca.2025.344661}},
  volume       = {{1379}},
  year         = {{2025}},
}