Dataset Information

Proteomic and lipidomic profiling of immune cell-derived subpopulations of extracellular vesicles

ABSTRACT: Introduction: Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles released by cells. They play important roles in intercellular communication and contribute to several physiological and pathological processes. Cells release subpopulations of EVs with distinct biogenesis and functions, however, we currently have few markers to differentiate them. This study therefore aimed to determine proteomic and lipidomic differences among four EV subpopulations of varying sizes and densities. Methods: Large and small EVs (L-EVs and S-EVs) were isolated from two immune cell lines by differential ultracentrifugation at 16,500 × g and 118,000 × g, respectively. The crude EVs were then further separated by density cushion centrifugation. EVs were isolated from the interphase between 1.079-1.146 and 1.146-1.185 g/ml, hereafter referred to as low density (LD) and high density (HD), respectively. This resulted in four subpopulations of EVs, namely, L-EV LD, L-EV HD, S-EV LD, and S-EV HD. The purity, morphology, size, and yield of EVs were determined by nanoparticle tracking analysis, electron microscopy, and western blot. The proteome and lipidome of the four subpopulations of EVs were determined with mass spectrometry. Results: A total of 5364 proteins were quantified in the dataset. L-EV and S-EVs as well as LD and HD were well separated. Briefly, L-EVs LD were enriched in mitochondrial proteins such as the TIMM/TOMM complex, MICOS, and ATP5 proteins, while L-EVs HD were enriched in proteins associated with the cytoskeleton, such as KIF proteins. Furthermore, S-EVs LD were enriched in tetraspanins and ESCRT machinery proteins, while S-EVs HD were enriched in histones, CCT proteins, and proteins from the complement pathway. While proteins such as flotillins, RABs, annexins, and integrins were enriched in two or several subpopulations. Our study quantified 108 lipids, and the most abundant lipids in EVs were phosphatidylcholine (PC), sphingomyelin, and phosphatidylethanolamine (PE). The most profound difference was that PE was less abundant in L-EVs LD as compared to the other EV subtypes and ceramides were enriched in L-EVs as compared to S-EVs. Conclusion: This study demonstrates that the proteome strongly differs in EV subpopulations separated at different densities. Furthermore, it validates several protein groups that have previously been suggested to be enriched in either S-EVs or L-EVs.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Proteomics Core Facility

LAB HEAD: Cecilia Lässer

PROVIDER: PXD063334 | Pride | 2026-03-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	4417_set1and2_SPS50_Fusion_220602_10to26_32to48.msf	Msf
	4417_set1and2_SPS50_Fusion_220602_10to26_32to48.pdResult	Other
	Fusion_220602_10.raw	Raw
	Fusion_220602_11.raw	Raw
	Fusion_220602_12.raw	Raw

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Publications

Proteomic and Lipidomic Profiling of Immune Cell-Derived Subpopulations of Extracellular Vesicles.

Lischnig Anna A Karimi Nasibeh N Larsson Per P Ekström Karin K Crescitelli Rossella R Olin Anna-Carin AC Lässer Cecilia C

Proteomics 20260110

Extracellular vesicles (EVs) are heterogeneous and play important roles in intercellular communication, contributing to physiological and pathological processes. Since few markers currently exist to differentiate subtypes of EVs, this study aimed to determine proteomic and lipidomic differences among four EV subpopulations. Large and small EVs (L-EVs and S-EVs) were isolated from human mast cells (HMC-1) and monocytes (THP-1) by differential ultracentrifugation and then further separated by dens ...[more]

PMID: 41518059

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Project description:Background: Huntington’s disease (HD) is an inherited autosomal dominant disorder characterized by progressive degeneration of the striatum and cerebral cortex, caused by a mutation in the huntingtin gene, which includes an aberrant expansion of a CAG repeat. The mutant huntingtin protein accumulates in endosomal compartments and alters the endosomal-lysosomal system, which can affect the production, secretion, and content of exosomes - extracellular vesicles (EVs) with an endosomal origin. Therefore, we investigated whether the levels and content of small EV subpopulations, including exosomes, are altered in the brains of HD patients. Methods: We isolated and characterized the proteome of two subtypes of small EVs from the striatum and cortex of HD brains with early or advanced degeneration, as well as from control brains, using differential ultracentrifugation followed by a high-resolution iodixanol density gradient and mass spectrometry analysis. Additionally, we explored the secretion of EVs by fibroblasts from HD patients compared to controls. Results: Our data indicate that the level of Annexin A2-enriched EVs increased in the HD cortex, whereas the level of Alix-enriched EVs was reduced in both the HD striatum and cortex compared to controls. In terms of content per EV, we found that the level of Annexin A2 and Alix were increased and reduced in HD, respectively. This altered cargo of Alix in EVs paralleled a progressive decrease in Alix protein in the cortex and striatum of HD patients, which correlated with the neuropathological state of the brain. Furthermore, in vitro, HD fibroblasts secreted EVs that were less enriched in Alix as compared to control fibroblasts, despite showing similar Alix protein levels in the cell lysates. Conclusions: Taken together, our data provide new insights into the pathophysiology of brain EVs in HD, identifying Alix as a novel marker of neuropathology. This opens new avenues to explore Alix as a potential biomarker of disease progression in peripheral tissues.