Browsing by Subject "pathway analysis"
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Item Identification of novel alternative splicing biomarkers for breast cancer with LC/MS/MS and RNA-Seq(BioMed Central Ltd., 2020-12-03) Zhang, Fan; Deng, Chris K.; Wang, Mu; Deng, Bin; Barber, Robert C.; Huang, GangBackground: Alternative splicing isoforms have been reported as a new and robust class of diagnostic biomarkers. Over 95% of human genes are estimated to be alternatively spliced as a powerful means of producing functionally diverse proteins from a single gene. The emergence of next-generation sequencing technologies, especially RNA-seq, provides novel insights into large-scale detection and analysis of alternative splicing at the transcriptional level. Advances in Proteomic Technologies such as liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), have shown tremendous power for the parallel characterization of large amount of proteins in biological samples. Although poor correspondence has been generally found from previous qualitative comparative analysis between proteomics and microarray data, significantly higher degrees of correlation have been observed at the level of exon. Combining protein and RNA data by searching LC-MS/MS data against a customized protein database from RNA-Seq may produce a subset of alternatively spliced protein isoform candidates that have higher confidence. Results: We developed a bioinformatics workflow to discover alternative splicing biomarkers from LC-MS/MS using RNA-Seq. First, we retrieved high confident, novel alternative splicing biomarkers from the breast cancer RNA-Seq database. Then, we translated these sequences into in silico Isoform Junction Peptides, and created a customized alternative splicing database for MS searching. Lastly, we ran the Open Mass spectrometry Search Algorithm against the customized alternative splicing database with breast cancer plasma proteome. Twenty six alternative splicing biomarker peptides with one single intron event and one exon skipping event were identified. Further interpretation of biological pathways with our Integrated Pathway Analysis Database showed that these 26 peptides are associated with Cancer, Signaling, Metabolism, Regulation, Immune System and Hemostasis pathways, which are consistent with the 256 alternative splicing biomarkers from the RNA-Seq. Conclusions: This paper presents a bioinformatics workflow for using RNA-seq data to discover novel alternative splicing biomarkers from the breast cancer proteome. As a complement to synthetic alternative splicing database technique for alternative splicing identification, this method combines the advantages of two platforms: mass spectrometry and next generation sequencing and can help identify potentially highly sample-specific alternative splicing isoform biomarkers at early-stage of cancer.Item Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma(MDPI, 2023-08-26) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloElevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.