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De novo identification of the blood plasma and serum peptides from samples of healthy donors

ABSTRACT: Previously, we published a dataset of human blood plasma and serum samples of 10 healthy males and 10 healthy females, fractionated on a set of sorbents (cation exchange Toyopearl CM-650M, CM Bio-Gel A, SP Sephadex C-25 and anion exchange QAE Sephadex A-25) and analyzed by LC-MS/MS individually and pooled in equal amounts (Supplementary Table S1, Sheet 1) [33]. Blood is a complex tissue and can theoretically contain products of all processes occurring in different parts of the body. To identify potential sources of “alien” (non-human) plasma peptides, we performed a de novo analysis of mass spectrometry data. De novo analysis is a less efficient method of identification than search against databases, since the accuracy and resolution of modern mass spectrometers such as QTOF or Orbitrap remains not high enough. However, we use de novo analysis to identify organisms that include the most abundant components of the complex peptide mixture. This procedure allows us to develop a hypothesis and then perform a standard search against a database of proteins of identified organisms. The de novo identification was carried out using PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Canada) and mass spectrometry driven BLAST analysis against the RefSeq non-redundant database NCBInr (https://www.ncbi.nlm.nih.gov/protein/). Mass spectra identified as fragments of proteins from different organisms were assigned to a taxonomic level at which these different organisms converged (Supplementary Table S2). 33. Arapidi, G. et al. Peptidomics dataset: Blood plasma and serum samples of healthy donors fractionated on a set of chromatography sorbents. Data Brief 18, 1204–1211 (2018).

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Blood Serum

DISEASE(S): Disease Free

SUBMITTER: Georgij Arapidi

LAB HEAD: Georgij Pavlovich Arapidi

PROVIDER: PXD027701 | Pride | 2024-06-14

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	4_PEAKS_de-novo.z01	Other
	4_PEAKS_de-novo.zip	Other
	I305_20140905_SWP100_CMBiogel20_B1_5-40_120min_7uLinj_1.wiff	Wiff
	I305_20140905_SWP100_CMBiogel20_B1_5-40_120min_7uLinj_1.wiff.scan	Wiff
	I305_MGFPeaklist.mgf	Mgf

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

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De novo identification of the blood plasma and serum peptides from samples of healthy donors

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