Dataset Information

Human serum extracellular vesicle proteomic profile depends on the enrichment method employed

ABSTRACT: The proteomic profiling of serum samples supposes a challenge due to the large abundance of few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, to enrich less abundant proteins, getting rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins, require specific strategies. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size exclusion chromatography, and two commercial precipitation methods based on different mechanism of action. To improve the performance of the techniques and promote purity of the preparations we have employed samples of 80 ul. The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yields a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. Size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit had more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriched differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mikel Azkargorta

LAB HEAD: Felix Elortza

PROVIDER: PXD029015 | Pride | 2022-02-17

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	10_UC2_12_Slot1-29_3-14-2020_7493.d.7z	Other
	11_UC3_13_Slot1-30_3-14-2020_7495.d.7z	Other
	12_UC4_14_Slot1-31_3-14-2020_7497.d.7z	Other
	13_SEC03_1_Slot1-32_3-15-2020_7500.d.7z	Other
	14_SEC13_15_Slot1-33_3-15-2020_7502.d.7z	Other

Items per page:

1 - 5 of 20

Publications

Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed.

Azkargorta Mikel M Iloro Ibon I Escobes Iraide I Cabrera Diana D Falcon-Perez Juan M JM Elortza Felix F Royo Felix F

International journal of molecular sciences 20211015 20

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promisin ...[more]

PMID: 34681804

Similar Datasets

Project description:Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel virus of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of host genomic DNA (hgDNA) contamination. Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA but are cell membrane-impermeable and thus are unable to bind protected DNA and RNA such as viral genomic material. DNA modified by EMA or PMA is not amplifiable by polymerase. In order to assess the capacity of EMA or PMA to lower hgDNA, serum or lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with different combination of treatment: with or without ultracentrifugation and incubation with or without different concentration of EMA or PMA. PVDA and HTS were used to evaluate the capacity of both techniques to detect the presence of PRRSV from each sample. Negative results were obtained by PVDA and low amount of PRRSV specific reads were obtained by HTS with untreated samples or samples treated only by ultracentrifugation. An increase capacity of PRRSV detection was observable by PVDA in EMA and PMA treated samples but PVDA best results were obtained following PMA treatment, with or without ultracentrifugation. HTS sensitivity was also improved by a treatment with EMA or PMA, but the number of reads was significantly higher in PMA treated samples. These results support the use of PMA as a treatment to increase sensitivity of PVDA and HTS. A non specific DNA probe is used as a negative hybridization control. Also, specifics probes targeting pUC19 plasmid DNA is used as a DNA array positive control as well as a localization control. Finally, there are 34 probes of 70 nucleotides spotted in duplicate were selected to target PRRSV conserved regions. A total of 80 different tests were done by DNA array in order to evaluate different treatments conditions with EMA or PMA, with or without ultracentrifugation. A total of 58 samples were tested on specific lung tissue homogenates spiked with different concentration of PRRSV and 12 samples were serum samples spiked with one concentration of PRRSV. Finally. A total of six samples