Dataset Information

Exome-based proteogenomics of HEK-293 human cell line: coding genomic variants identified at the level of shotgun proteome

ABSTRACT: Genomic and proteomic data were integrated into the proteogenomic workflow to identify coding genomic variants of Human Embryonic Kidney 293 (HEK-293) standard cell line at the proteome level. Shotgun proteome data published by Geiger et al (2012) and obtained in this work for HEK-293 were searched against the customized genomic databased generated using exome data published by Lin et al (2014). 54 unique variants out of ~1,200 coding variants annotated in the exome were found at the proteome level. 27 of them were validated by two search engines, X!Tandem and Andromeda. 16 (60%) of those validated variants were confidently identified in both own and published proteome datasets. Some of the variants found belonged to widely known genomic polymorphisms originated from the germline, while others are more likely to result from somatic mutations. Notably, the peptide subsets identified by only one, or the other search engine were enriched by the sequences with miscleavages. This can be due to the large presence of false-positive hits in these subsets that is especially true for the subset of variant peptides. High-resolution mass-spectra of HEK-293 cell line were deposited to ProteomeXchange repository, project accession PXD002613.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell, Kidney

SUBMITTER: Irina Ilina

LAB HEAD: Sergei Moshkovskii

PROVIDER: PXD002613 | Pride | 2016-07-22

REPOSITORIES: Pride

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Publications

Exome-based proteogenomics of HEK-293 human cell line: Coding genomic variants identified at the level of shotgun proteome.

Lobas Anna A AA Karpov Dmitry S DS Kopylov Arthur T AT Solovyeva Elizaveta M EM Ivanov Mark V MV Ilina Irina Y IY Lazarev Vassily N VN Kuznetsova Ksenia G KG Ilgisonis Ekaterina V EV Zgoda Victor G VG Gorshkov Mikhail V MV Moshkovskii Sergei A SA

Proteomics 20160621 14

Genomic and proteomic data were integrated into the proteogenomic workflow to identify coding genomic variants of Human Embryonic Kidney 293 (HEK-293) cell line at the proteome level. Shotgun proteome data published by Geiger et al. (2012), Chick et al. (2015), and obtained in this work for HEK-293 were searched against the customized genomic database generated using exome data published by Lin et al. (2014). Overall, 112 unique variants were identified at the proteome level out of ∼1200 coding ...[more]

PMID: 27233776

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Project description:MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486. The hearts of 3 male 8 month old Sprague-Dawley rats were rapidly extracted after euthanasia with sodium pentobarbital. A section of the free wall of the left ventricle was dissected into epicardium, mid-myocardium and endocardium by cutting approximately 1 mm from the epicardial and endocardial surfaces. Small RNA was extracted (miRNeasy Kit; Qiagen, Crawley UK), quantified (Nanodrop; Thermo Scientific) and quality assessed for degradation (RNA Nano Chip, Bioanalyser 2100; Aligent Technologies, Wokingham UK; only samples with a RNA integrity no. (RIN) ≥8 were carried forward) and retention of small RNA (Small RNA Chip, Bioanalyser 2100). Small RNA was preferentially ligated with adapters, reverse transcribed into cDNA and amplified with 9 individually tagged primer indices (TruSeq Small RNA Sample Preparation Kit; Illumina, Little Chesterford, UK) and a library of small RNA created for each sample. After gel purification the cDNA products were again analysed on the bioanalyser using a High Sensitivity DNA Chip and assessed for the presence and concentration of the peak corresponding to ligated and tagged miRNA (approximately 147nt). Only samples with suitable RIN values exhibiting good retention of small RNA species were used for library preparation. After pooling, the samples were sequenced by TrinSeq (Trinity Genome Sequencing Lab & Neuropsychiatric Genetics Group, Trinity College Dublin, Ireland (http://www.medicine.tcd.ie/sequencing); using TruSeq SR Cluster Kit v5 (Illumina) and the resultant data trimmed and aligned to miRBase v18 (CLC Genomics Workbench v4.0; CLC bio, Swansea UK).

Dataset Information

Exome-based proteogenomics of HEK-293 human cell line: coding genomic variants identified at the level of shotgun proteome

Publications

Exome-based proteogenomics of HEK-293 human cell line: Coding genomic variants identified at the level of shotgun proteome.

Similar Datasets

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