Proteome of Gastrointestinal Stromal Tumor-Derived Exosomes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Safinur Atay, PhD
PROVIDER: MSV000081625 | MassIVE | Wed Oct 18 15:00:00 BST 2017
SECONDARY ACCESSION(S): PXD007997
REPOSITORIES: MassIVE
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Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.
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Project description:The raw files of the proteomics dataset are N=18 and a brief description of the files is shown below:
AF1 raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 1
AF2.raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 2
AFA1.raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 3
AFA2.raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 4
AFB1.raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 5
AFB2.raw: exosomes derived from Amniotic Fluid-Mesenchymal Stromal cells 6
HL1A.raw: exosomes derived from Hepatocyte like cells 1
HL1B.raw: exosomes derived from Hepatocyte like cells 2
HL2A.raw: exosomes derived from Hepatocyte like cells 3
HL2B.raw: exosomes derived from Hepatocyte like cells 4
HL3A.raw: exosomes derived from Hepatocyte like cells 5
HL3B.raw: exosomes derived from Hepatocyte like cells 6
HPLA1.raw: exosomes derived from Hepatic Progenitor-like cells 1
HPLA2.raw: exosomes derived from Hepatic Progenitor-like cells 2
HPLB1.raw: exosomes derived from Hepatic Progenitor-like cells 3
HPLB2.raw: exosomes derived from Hepatic Progenitor-like cells 4
HPLA.raw: exosomes derived from Hepatic Progenitor-like cells 5
HPLB.raw: exosomes derived from Hepatic Progenitor-like cells 6
Raw files analysis was performed with Proteome Discoverer 1.4 (Thermo) software package, using the Sequest search engine and the Uniprot human (Homo sapiens) reviewed database, downloaded on December 15, 2017, including 20,243 entries. The search was performed using carbamidomethylation of cysteine as static and oxidation of methionine as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05 Da were allowed. False discovery rate (FDR) validation was based on q value: target FDR : 0.05. Label free quantification was performed by utilizing the precursor ion area values exported from the total ion chromatogram as defined by the Proteome Discoverer v. 1.4.0.288 (Thermo Scientific). Output files from Proteome Discoverer were processed with R programming language for statistical computing (version 4.0.3).
The following comparisons were made: AF vs HL, AF vs HPL and HL vs HPL.
The msf files are N=18 and have the same name as the raw files above.
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