Data on corneal proteome and differentially expressed corneal proteins in highly myopic chicks using a data independent quantification approach.
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ABSTRACT:
SUBMITTER: Kang BS
PROVIDER: S-EPMC6811982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The retina plays an important regulatory role in ocular growth. To screen for new retinal candidate genes that could be involved in the inhibition of ocular growth, we used chick microarrays to analyze the changes in retinal mRNA expression after myopic defocus was imposed by positive lens-wear. Chicks were raised under a 12h-light/12h-dark cycle (light-onset: 8:00 am and light-offset: 8:00 pm) with unrestricted access to water and food. On the day prior to the experiment, velcro rings were glued to the feathers around the eye under diethylether anaesthesia. Four male white leghorn chicks, aged 9 days, wore +6.9D spectacle lenses over both eyes for 24 hours. Four untreated age-matched male chicks from the same batch served as controls. After decapitation of the chicks, the eyes were enucleated and the retina was prepared. The retinae from both eyes of each chick were pooled for RNA isolation. Keywords: gene expression comparison
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Project description:High-throughput data-independent acquisition (DIA) is the method of choice for quantitative proteomics, combining the best practices of targeted and shotgun approaches. The resultant DIA spectra are, however, highly convolved and with no direct precursor-fragment correspondence, complicating biological sample analysis. Here, we present CANDIA (canonical decomposition of data-independent-acquired spectra), a GPU-powered unsupervised multiway factor analysis framework that deconvolves multispectral scans to individual analyte spectra, chromatographic profiles, and sample abundances, using parallel factor analysis. The deconvolved spectra can be annotated with traditional database search engines or used as high-quality input for de novo sequencing methods. We demonstrate that spectral libraries generated with CANDIA substantially reduce the false discovery rate underlying the validation of spectral quantification. CANDIA covers up to 33 times more total ion current than library-based approaches, which typically use less than 5% of total recorded ions, thus allowing quantification and identification of signals from unexplored DIA spectra.
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Project description:Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS(2) spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS(1)-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches.
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Project description:PURPOSE: The retina plays an important regulatory role in ocular growth. To screen for new retinal candidate genes that could be involved in the inhibition of ocular growth, we used chick microarrays to analyze the changes in retinal mRNA expression after myopic defocus was imposed by positive lens wear. METHODS: Four male white leghorn chicks, aged nine days, wore +6.9D spectacle lenses over both eyes for 24 h. Four untreated age-matched male chicks from the same batch served as controls. The chicks were euthanized, and retinas from both eyes of each chick were pooled. RNA was isolated and labeled cRNA was prepared. These samples were hybridized to Affymetrix GeneChip Chicken Genome arrays with more than 28,000 characterized genes. After comparison of multiple normalization methods, GC-RMA and a false-discovery rate of 6% was chosen for normalization of the data. The expression of 16 candidate genes was further studied, using semiquantitative real-time RT-PCR. In addition, the expression of the mRNA of some of these candidate genes was assessed in chicks that wore either +6.9D lenses for 4 h or -7D lenses for 24 h. RESULTS: 123 transcripts were found to be differentially expressed (p<0.05; at least 1.5-fold change in expression level), with an absolute mean fold-change of 1.97+/-1.16 (mean+/-standard deviation). Nine of the sixteen genes that were examined by real-time RT-PCR were validated. Regardless of whether positive or negative lenses were worn, six of these nine genes were regulated in the same direction after 24 h: arginyltransferase 1 (ATE1), E74-like factor 1 (ELF1), growth factor receptor-bound protein 2 (GRB2), SHQ1 homolog (S. cerevisiae) (SHQ1), spectrin, beta, non-erythrocytic 1 (SPTBN1), prepro-urotensin II-related peptide (pp-URP). Three genes responded differently to positive and negative lens treatment after 24 h: ATP-binding cassette, sub-family C, member 10 (ABCC10), CD226 molecule (CD226) and oxysterol binding protein 2 (OSBP2). CONCLUSIONS: The validated genes that were regulated only by myopic defocus may represent elements in a pathway generating a "stop-signal" for eye growth. Some of the genes identified in this study have so far not been described in the retina. Further investigation of their function may improve the understanding of the signaling cascades in emmetropization. More general, published microarray data are variable among different animal models (mouse, chick, monkeys), tissues (retina, retina/retinal pigment epithelium), treatments (diffusers, lenses, lid-suture), as well as different treatment durations (hours, days), and comparisons remain difficult. That only a small number of common genes were found emphasizes the need for careful normalization of the experimental parameters.
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