PXD047505

PXD047505 is an original dataset announced via ProteomeXchange.

Title	Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles
Description	The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is therefore to always perform HI of FBS prior to EV depletion.
HostingRepository	PRIDE
AnnounceDate	2024-01-26
AnnouncementXML	Submission_2024-01-26_06:05:23.306.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Johannes Fuchs
SpeciesList	scientific name: Bos taurus (Bovine); NCBI TaxID: 9913;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-12-04 06:11:24	ID requested
⏵ 1	2024-01-26 06:05:24	announced

10.1002/jev2.12408;

Urz, ì O, Bergqvist M, L, ä, sser C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R, Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. J Extracell Vesicles, 13(1):e12408(2024) [pubmed]

submitter keyword: EV depletion by ultracentrifugation, foetal bovine serum (FBS),extracellular vesicles, FBS heat-inactivation

Rossella Crescitelli
contact affiliation	Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
contact email	rossella.crescitelli@gu.se
lab head
Johannes Fuchs
contact affiliation	Gothenburg University
contact email	johannes.fuchs@gu.se
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD047505
     1. Label: PRIDE project
     2. Name: Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles